Vascular endothelial growth factor receptor targeting peptide-elastin fusion polypeptides

ABSTRACT

Disclosed is a fusion polypeptide for inhibiting neovascularization, including a peptide specifically binding to vascular endothelial growth factor (VEGF) receptors, and a hydrophilic elastin-based polypeptide (hydrophilic EBP) linked to the peptide.

CROSS-REFERENCE TO RELATED APPLICATION

This is a divisional application of co-pending U.S. application Ser. No.15/744,278, filed on Jan. 12, 2018, which was a national stage of PCTapplication No. PCT/KR2016/011757, filed on Oct. 19, 2016, and claimspriority to and the benefit of Korean Patent Applications Nos.10-2016-0042655 and 10-2016-0135510, filed on Apr. 7, 2016 and Oct. 19,2016, respectively, the disclosures of which are incorporated herein byreference in its entirety.

SEQUENCE LISTING

This divisional application contains a Sequence Listing submitted viaEFS-Web and hereby incorporated by reference in its entirety. TheSequence Listing is named DAH-266NPD1-2A_Sequence_Listing_Revised.txt,created on Jun. 25, 2020, and 66,430 bytes in size.

BACKGROUND 1. Field of the Invention

The present invention relates to a fusion polypeptide and aself-assembled nanostructure for inhibiting neovascularization, and moreparticularly, to a fusion polypeptide for inhibiting neovascularizationincluding a peptide specifically binding to vascular endothelial growthfactor (VEGF) receptors; and a hydrophilic elastin-based polypeptide(hydrophilic EBP) linked to the peptide.

In addition, the present invention relates to a fusion polypeptide forinhibiting neovascularization including a peptide specifically bindingto VEGF receptors; a hydrophilic EBP linked to the peptide; and ahydrophobic elastin-based polypeptide (hydrophobic EBP) linked to thehydrophilic EBP, and a self-assembled nanostructure thereof.

2. Discussion of Related Art

Peptides, polypeptides and proteins having specific functions, such ascell penetration, cell attachment, binding affinity to target molecules,therapeutic efficacy, and site-specific conjugation, may be fusedtogether to form a multifunctional artificial chimera or fusion proteinsuitable for smart drug delivery systems.

Such fusion proteins exhibit high specificity, high activity, longhalf-lives, low accumulation in certain organs, and low side effectswhen used in vivo. Recently, multifunctional fusion proteins have beenprepared at the genetic level with recombinant DNA technology and thefollowing are precisely regulated: (1) sequence and composition of aminoacids, (2) fusion order, (3) monodisperse molecular weight, (4)hydrophilicity and hydrophobicity, (5) environmental responsiveness, (6)biocompatibility and biodegradability, (7) toxicity and immunogenicity,(8) pharmacokinetics and pharmacodynamics.

The fusion proteins are expressed in high yield (0.1 to 0.5 g per literof culture) in prokaryotic or eukaryotic expression systems, and arepurified by column chromatography or inverse transition cycling (ITC)for analyzing unique phase transition behaviors induced bystimuli-responsiveness of the fusion proteins. For example,antimicrobial host-defense peptides were genetically fused withpolypeptide F4 to overcome limitations of the host-defense peptides,such as low stability, short half-lives and high production cost.Furthermore, tumor-targeting antibodies were prepared in mice using ahybridoma technology, and antigen-binding variable domains of mouseantibodies were combined with human IgG to reduce immunogenicity inpatients. A large number of artificial fusion proteins are inpreclinical and clinical development, and technologies usingmulti-functional artificial chimeric or fusion protein-basedtherapeutics are growing exponentially.

Elastin-based polypeptides (EBPs) are thermal response biopolymersderived from elastomeric domains. Elastin is a major protein componentof the extracellular matrix (ECM). EBPs are modified to have thermalsensitivity based on an elastomeric domain and are composed of apentapeptide repeat unit, Val-Pro-(Gly or Ala)-X_(aa)-Glys[VP (G orA)XG]. EBPs are thermally responsive polypeptides, and transitiontemperatures thereof are readily controlled to form nanostructures fordrug delivery.

X_(aa) is a guest residue and may be any amino acid except proline.Depending on a sequence corresponding to the repeat unit, there are twotypes of EBPs, one is an elastin-based polypeptide with elasticity(EBPE) with a sequence of Val-Pro-Gly-X_(aa)-Gly and the other is anelastin-based polypeptide with plasticity (EBPP) with a sequence ofVal-Pro-Ala-X_(aa)-Gly.

EBPs exhibit a lower critical solution temperature (LCST) behavior inwhich a reversible phase transition is observed depending ontemperature. The LCST provides the advantage of using an easypurification method such as inverse transition cycling (ITC) and theadvantage of being thermally triggered to self-assemble into particles,gels, fibers and other structures.

Diblocks composed of EBP blocks that have different sequences are usedto form self-assembled structures. An EBP diblock copolymer is composedof two EBPs, in which the EBPs have different sequences and differenttransition temperatures (T_(t)) to form a self-assembled micellarstructure. When the temperature of an EBP diblock copolymer solutionincreases above a lower T_(t), EBPs that have a low T_(t) becomeinsoluble whereas EBPs that have a high T_(t) are soluble, andamphiphilic diblock EBPs are self-assembled into micellar structures.EBP diblock copolymers may be fused with other functional peptides,e.g., a cell penetrating peptide capable of penetrating cells, to havefunctional multivalency as micellar structures.

Soluble EBPs may be used as inert protein-based biomaterials, likepoly(ethylene glycol) (PEG), and as drug delivery carriers with drugs orother functional proteins, for advanced drug delivery systems,regenerative medicine, and tissue engineering.

EBPs may be easily purified and have stimuli-triggered phasetransitions, allowing for genetic fusion with other functional proteinsand exploitation of the advantages of EBPs. For example, EBPs may befused with an interleukin-1 receptor antagonist (IL-1Ra) to create aninjectable drug reservoir for treating osteoarthritis.

In addition, with the advancement of therapeutic EBP fusion proteins,self-assembled micelles of EBP block copolymers are being studied. AnEBP diblock copolymer is composed of two different EBP blocks, each ofwhich has a different sequence, configuration and chain length, whichallows each EBP block to have a unique transition temperature (T_(t)).When temperature rises, the EBP block with a low T_(t) becomesinsoluble, while another EBP block with a high T_(t) becomes solubleabove the low T_(t). Due to the amphiphilic properties of the EBPdiblock copolymer above the low T_(t), the EBP diblock copolymerself-assembles into a core-shell micellar nanostructure. In addition,EBP diblock copolymers may be fused with other functional peptides orproteins to become functionally multivalent. Both the core and shell ofthe EBP micellar nanostructure may be used differently as drug deliverycarriers.

Recently, a considerable number of cancer-related diseases have beenknown to result from abnormal neovascularization in tumors.Physiological neovascularization in organisms is strictly regulated andis only activated under specific conditions. However, excessiveformation of blood vessels due to disruption of regulation may lead todiseases such as non-tumor diseases as well as cancers. Underphysiological conditions, including development, growth, wound healing,and regeneration, neovascularization is stimulated by vascularendothelial growth factor (VEGF). VEGF binds to two types of VEGFreceptors (VEGFRs), including VEGFR1 (fms-like tyrosine kinase-1 orFlt1) and VEGFR2 (kinase insert domain-containing receptor orFlk-1/KDR), present on cell membranes. Selective binding of VEGF to VEGFreceptors delivers a growth signal to vascular endothelial cells, whichin turn triggers neovascularization.

Therefore, to inhibit neovascularization in various diseases such astumor growth, cancer metastasis, retinal neovascularization, cornealneovascularization, diabetic retinopathy and asthma, various strategiesfor anti-neovascularization have been employed. In particular,anti-neovascularization strategies for treatment of ocularneovascularization include initiating a signal that inhibitsneovascularization using neovascularization inhibitors such as pigmentepithelial-derived factor (PEDF) and caffeic acid (CA), and blockingneovascularization signals by interfering with binding of VEGF toreceptors thereof (VEGFRs). In particular, since biomacromolecules andtargeting peptides have high affinity for VEGFR1 and competitively bindto VEGFR1 as receptor antagonists, using biomacromolecules or targetingpeptides as antibodies may be a challenging strategy related tointerfering with binding of VEGF to VEGF receptors.

An anti-Flt1 peptide identified by PS-SPCL (positionalscanning-synthetic peptide combinatorial library) screening, one amonghigh throughput screening (HTS) systems, is a hexa-peptide having anamino acid sequence of Gly-Asn-Gln-Trp-Phe-Ile (GNQWFI). The anti-Flt1peptide, as a VEGFR1-specific antagonist, specifically binds to VEGFR1,which prevents VEGFR1 from interacting with all VEGFR1 ligands,including placental growth factor (PIGF), as well as VEGF. To increasethe half-life of the anti-Flt1 peptide in vivo, anti-Flt1peptide-hyaluronate (HA) conjugates have been studied in connection withthe formation of self-assembled micelle structures which encapsulategenistein, dexamethasone or tyrosine-specific protein kinase inhibitors.Although conjugation of the anti-Flt1 peptide with HA polymers increasesthe half-life of the anti-Flt1 peptide in the body, conjugationefficiency and micellar structures are heterogeneous due to polydisperseHA polymer molecular weights, random distribution, and inconsistency ofconjugation efficiency of the anti-Flt1 peptides and the HAs.

The present inventors have continued to study fusion polypeptides forinhibiting neovascularization. As a result, a novel fusion polypeptidein which a peptide targeting vascular endothelial growth factor (VEGF)receptors and an elastin-based polypeptide were fused was developed andthe present invention was completed.

SUMMARY OF THE INVENTION

Therefore, the present invention has been made in view of the aboveproblems, and it is an objective of the present invention to provide anovel fusion polypeptide for inhibiting neovascularization.

It is another objective of the present invention to provide aself-assembled nanostructure of the fusion polypeptide.

It is still another objective of the present invention to provide acomposition for treating diseases caused by neovascularization.

It is yet another objective of the present invention to provide a methodof inhibiting neovascularization in individuals.

According to an aspect of the present invention, there is provided afusion polypeptide for inhibiting neovascularization, including:

a peptide specifically binding to vascular endothelial growth factor(VEGF) receptors; and

a hydrophilic elastin-based polypeptide (hydrophilic EBP) linked to thepeptide.

The peptide specifically binding to VEGF receptors may be a peptide thatspecifically binds to VEGF receptor Flt1 or Flk-1/KDR.

The peptide specifically binding to VEGF receptors may be a peptide thatspecifically binds to VEGF receptor Flt1 or Flk-1/KDR, and is alsocalled “VEGF receptor-specific peptide” or “VEGF receptor-targetingpeptide”. The VEGF receptor-specific peptide may be any of anti-Flt1 oranti-Flk-1/KDR (poly)peptides well known in the art. For example, thepeptide may be an anti-Flt1 peptide [SEQ ID NO. 38], but is necessarilylimited thereto.

The hydrophilic EBP may be composed of an amino acid sequencerepresented by Formula 1 or 2 below:

Formula 1 [SEQ ID NO. 1] n; or   Formula 2 [SEQ ID NO. 2] n, wherein  SEQ ID NO. 1 is consisted of [VPGXG VPGXG VPGXG VPGXG VPGXG VPGXG];  SEQ ID NO. 2 is consisted of [VPAXG VPAXG VPAXG VPAXG VPAXG VPAXG];

n is an integer of 1 or more, and represents the number of repeats ofSEQ ID NO. 1 or SEQ ID NO. 2; and

X is an amino acid other than proline, is selected from any natural orartificial amino acid when the pentapeptide VPGXG or VPAXG is repeated,and at least one of X is a hydrophilic amino acid.

The hydrophilic EBP may be composed of an amino acid sequencerepresented by Formula 1 or 2 below:

in Formula 1, n is 1, each X of the pentapeptide repeats is consistedof,

A (Ala), G (Gly), and I (Ile) in a ratio of 1:4:1 [SEQ ID NO. 20];

K (Lys), G (Gly), and I (Ile) in a ratio of 1:4:1 [SEQ ID NO. 22];

D (Asp), G (Gly), and I (Ile) in a ratio of 1:4:1 [SEQ ID NO. 24]; or

E (Glu), G (Gly), and I (Ile) in a ratio of 1:4:1 [SEQ ID NO. 26],

or

in Formula 2, n is 1, and the pentapeptide repeats

in Formula 2, n is 1, and each X of the pentapeptide repeats isconsisted of,

A (Ala), G (Gly), and I (Ile) in a ratio of 1:4:1 [SEQ ID NO. 21];

K (Lys), G (Gly), and I (Ile) in a ratio of 1:4:1 [SEQ ID NO. 23];

D (Asp), G (Gly), and I (Ile) in a ratio of 1:4:1 [SEQ ID NO. 25]; or

E (Glu), G (Gly), and I (Ile) in a ratio of 1:4:1 [SEQ ID NO. 27].

The hydrophilic EBP may include an amino acid sequence represented byFormula 2 below:

in Formula 2, n is 3, 6, 12 or 24, and the pentapeptide repeats

correspond to SEQ ID NO. 41, SEQ ID NO. 42, SEQ ID NO. 43 or SEQ ID NO.44 and each X of the pentapeptide repeats is composed of A (Ala), G(Gly), and I (Ile) in a ratio of 1:4:1 or

in Formula 2, n is 12, and the pentapeptide repeats

correspond to [SEQ ID NO. 45] and each X of the pentapeptide repeats iscomposed of E (Glu), G (Gly), and I (Ile) in a ratio of 1:4:1.

The fusion polypeptide according to the present invention may furtherinclude a hydrophobic elastin-based polypeptide (hydrophobic EBP) linkedto the hydrophilic EBP.

That is, the fusion polypeptide may include of the following:

a peptide specifically binding to vascular endothelial growth factor(VEGF) receptors;

a hydrophilic elastin-based polypeptide (hydrophilic EBP) linked to thepeptide; and

a hydrophobic elastin-based polypeptide (hydrophobic EBP) linked to thehydrophilic EBP.

The hydrophobic EBP may include an amino acid sequence represented byFormula 1 or 2 below:

Formula 1 [SEQ ID NO. 1] n; or   Formula 2 [SEQ ID NO. 2] n, wherein  SEQ ID NO. 1 is consisted of [VPGXG VPGXG VPGXG VPGXG VPGXG VPGXG];  SEQ ID NO. 2 is consisted of [VPAXG VPAXG VPAXG VPAXG VPAXG VPAXG];

n is an integer of 1 or more, and represents the number of repeats ofSEQ ID NO. 1 or SEQ ID NO. 2; and

X is an amino acid other than proline, is selected from any natural orartificial amino acid when the pentapeptide VPGXG or VPAXG is repeated,and at least one of X is a hydrophobic or aliphatic amino acid.

The hydrophobic EBP may be consisted of an amino acid sequencerepresented by Formula 1 or 2 below:

in Formula 1, n is 1, and each X of the pentapeptide repeats isconsisted of,

G (Gly), A (Ala), and F (Phe) in a ratio of 1:3:2 [SEQ ID NO. 29];

K (Lys), A (Ala), and F (Phe) in a ratio of 1:3:2 [SEQ ID NO. 30];

D (Asp), A (Ala), and F (Phe) in a ratio of 1:3:2 [SEQ ID NO. 31];

K (Lys) and F (Phe) in a ratio of 3:3 [SEQ ID NO. 32];

D (Asp) and F (Phe) in a ratio of 3:3 [SEQ ID NO. 33];

H (His), A (Ala), and I (Ile) in a ratio of 3:2:1 [SEQ ID NO. 34];

H (His) and G (Gly) in a ratio of 5:1 [SEQ ID NO. 35]; or

G (Gly), C (Cys), and F (Phe) in a ratio of 1:3:2 [SEQ ID NO. 36].

The hydrophobic EBP may include an amino acid sequence represented byFormula 2 below:

in Formula 2, n is 12, and each X of the pentapeptide repeats isconsisted of G (Gly), A (Ala), and F (Phe) in a ratio of 1:3:2 [SEQ IDNO. 46], or

in Formula 2, n is 24, and each X of the pentapeptide repeats isconsisted of

G (Gly), A (Ala), and F (Phe) in a ratio of 1:3:2 [SEQ ID NO. 47].

In one embodiment, the fusion polypeptide of the present invention maybe composed of an amino acid sequence corresponding to SEQ ID NO. 48,SEQ ID NO. 49, SEQ ID NO. 50 or SEQ ID NO. 51. That is, the fusionpolypeptide may be represented as follows:

the fusion polypeptide includes,

an anti-Flt1 peptide; and a hydrophilic EBP [A₁G₄I₁]₃ linked to theanti-Flt1 peptide, and represented by [SEQ ID NO. 48];

an anti-Flt1 peptide; and a hydrophilic EBP [A₁G₄I₁]₆ linked to theanti-Flt1 peptide, and represented by [SEQ ID NO. 49];

an anti-Flt1 peptide; and a hydrophilic EBP [A₁G₄I₁]₁₂ linked to theanti-Flt1 peptide, and represented by [SEQ ID NO. 50]; or

an anti-Flt1 peptide; and a hydrophilic EBP [A₁G₄I₁]₂₄ linked to theanti-Flt1 peptide, and represented by [SEQ ID NO. 51].

In another embodiment, the fusion polypeptide of the present inventionmay include an amino acid sequence corresponding to SEQ ID NO. 52 or SEQID NO. 53. That is, the fusion polypeptide may be represented asfollows:

the fusion polypeptide includes,

an anti-Flt1 peptide; a hydrophilic EBP [E₁G₄I₁]₁₂; and a hydrophobicEBP [G₁A₃F₂]₁₂, and represented by [SEQ ID NO. 52]; or

an anti-Flt1 peptide; a hydrophilic EBP [E₁G₄I₁]₁₂; and a hydrophobicEBP [G₁A₃F₂]₂₄, and represented by [SEQ ID NO. 53].

According to the present invention, the fusion polypeptide composed of aVEGF receptor-specific peptide; a hydrophilic EBP; and a hydrophobic EBPmay form a self-assembled nanostructure having a core-shell structure,when the hydrophobic EBP forms a core structure and the hydrophilic EBPand the VEGF receptor-specific peptide form a shell structure by atemperature stimulus.

The self-assembled nanostructure may include a multivalent VEGFreceptor-specific peptide as a shell.

Specifically, an anti-Flt1 peptide which is a VEGF receptor-specificpeptide is exemplified. A fusion polypeptide composed of an anti-Flt1peptide; a hydrophilic EBP; and a hydrophobic EBP may form aself-assembled nanostructure having a core-shell structure, when thehydrophobic EBP forms a core structure and the hydrophilic EBP and theanti-Flt1 peptide form a shell structure by a temperature stimulus.

The self-assembled nanostructure may include a multivalent anti-Flt1peptide as a shell, which provides greatly enhanced binding affinity toVEGF receptors.

According to another aspect of the present invention, there is provideda composition for treating diseases caused by neovascularization,including the fusion polypeptide.

According to still another aspect of the present invention, there isprovided a method of inhibiting neovascularization in individuals,including a step of administering the therapeutic composition toindividuals.

When a fusion polypeptide composed of a VEGF receptor-specific peptide;and a hydrophilic EBP is exemplified, the VEGF receptor-specific peptideof the fusion polypeptide may be non-covalently bound to a VEGF receptorto inhibit neovascularization (FIGS. 5A to 5D).

In addition, an example of a fusion polypeptide composed of a VEGFreceptor-specific peptide; a hydrophilic EBP; and a hydrophobic EBP isas follows.

The fusion polypeptide may form a self-assembled nanostructure having acore-shell structure, when the hydrophobic EBP forms a core structureand the hydrophilic EBP and the VEGF receptor-specific peptide form ashell structure by a temperature stimulus, and

the self-assembled nanostructure may include a multivalent VEGFreceptor-specific peptide as a shell, whereby binding affinity betweenthe self-assembled nanostructure and a VEGF receptor increases, and VEGFfails to bind to the VEGF receptor, thereby inhibitingneovascularization.

The diseases caused by neovascularization may be any one or moreselected from the group comprising diabetic retinopathy, retinopathy ofprematurity, macular degeneration, choroidal neovascularization,neovascular glaucoma, eye diseases caused by corneal neovascularization,corneal transplant rejection, corneal edema, corneal opacity, cancer,hemangioma, hemangiofibroma, rheumatoid arthritis, and psoriasis, butare necessarily limited thereto.

The term “vascular endothelial growth factor (VEGF)” used in the presentinvention refers to a factor that stimulates new blood vessel formation.VEGF binds to VEGF receptors to deliver a growth signal to vascularendothelial cells, which in turn triggers neovascularization.

The fusion polypeptide of the present invention functions to preventVEGF from binding to VEGF receptors.

The term “amino acid” used in the present invention refers to a naturalor artificial amino acid, preferably a natural amino acid. For example,the amino acid includes glycine, alanine, serine, valine, leucine,isoleucine, methionine, glutamine, asparagine, cysteine, histidine,phenylalanine, arginine, tyrosine, tryptophan and the like.

The properties of these amino acids are well known in the art.Specifically an amino acid exhibits hydrophilicity (negative or positivecharge) or hydrophobicity, and also exhibits aliphatic or aromaticproperties.

As used herein, abbreviations such as Gly (G) and Ala (A) are amino acidabbreviations. Gly is an abbreviation for glycine, and Ala is anabbreviation for alanine. In addition, glycine is represented by G andalanine by A. The abbreviations are widely used in the art.

In the present invention, “hydrophilic amino acid” is an amino acidexhibiting hydrophilic properties, and includes lysine, arginine and thelike.

In addition, “hydrophobic amino acid” is an amino acid exhibitinghydrophobic properties, and includes phenylalanine, leucine and thelike.

The term “polypeptide” used herein refers to any polymer chain composedof amino acids. The terms “peptide” and “protein” may be usedinterchangeably with the term polypeptide, and also refer to a polymerchain composed of amino acids. The term “polypeptide” includes naturalor synthetic proteins, protein fragments and polypeptide analogs havingprotein sequences. A polypeptide may be a monomer or polymer.

The term “phase transition” refers to a change in the state of amaterial, such as when water turns into water vapor or ice turns intowater.

The polypeptide according to the present invention is basically anelastin-based polypeptide (EBP) with stimuli-responsiveness. The“elastin-based polypeptide” is also called “elastin-like polypeptide(ELP)”. The term is widely used in the technical field of the presentinvention.

In the present specification, X_(aa) (or X) refers to a “guest residue”.Various types of EBPs according to the present invention may be preparedby variously introducing X_(aa).

EBP undergoes a reversible phase transition at a lower critical solutiontemperature (LCST), also referred to as a transition temperature(T_(t)). EBPs are highly water-soluble below T_(t), but become insolublewhen temperature exceeds T_(t).

In the present invention, the physicochemical properties of EBPs aremainly controlled by combination of a pentapeptide repeat unitVal-Pro-(Gly or Ala)-X_(aa)-Gly. Specifically, the third amino acid ofthe repeat unit is responsible for determining the relative mechanicalproperties of the EBPs. For example, according to the present invention,the third amino acid Gly is responsible for determining elasticity, orAla is responsible for determining plasticity. Elasticity and plasticityare properties that appear after a phase transition occurs.

In addition, the hydrophobicity of a guest residue X_(aa), the fourthamino acid, and multimerization of a pentapeptide repeat unit all affectT_(t).

The EBP according to the present invention may be a polypeptide composedof pentapeptide repeats, and a polypeptide block, i.e., an EBP block,may be formed when the polypeptide is repeated. Specifically, ahydrophilic or hydrophobic EBP block may be formed. The hydrophilic orhydrophobic properties of an EBP block according to the presentinvention are closely related to the transition temperature of the EBP.

The transition temperature of the EBP is also determined by the aminoacid sequence of the EBP and the molecular weight thereof. A number ofstudies on the relationship between an EBP sequence and T_(t) have beenconducted by Urry et al (see Urry D. W., Luan C.-H., Parker T. M., GowdaD. C., Parasad K. U., Reid M. C., and Safavy A. 1991. TEMPERATURE OFPOLYPEPTIDE INVERSE TEMPERATURE TRANSITION DEPENDS ON MEAN RESIDUEHYDROPHOBICITY. J. Am. Chem. Soc. 113: 4346-4348.). According to theabove reference, when, in a pentapeptide of Val-Pro-Gly-Val-Gly, thefourth amino acid, a “guest residue”, is replaced with a residue that ismore hydrophilic than Val, T_(t) is increased compared to the originalsequence. On the other hand, when the guest residue is replaced with aresidue that is more hydrophobic than Val, T_(t) is decreased comparedto the original sequence. That is, it was found that a hydrophilic EBPhas a high T_(t) and a hydrophobic EBP has a relatively low T_(t). Basedon these findings, it has become possible to prepare an EBP having aspecific T_(t) by determining which amino acid is used as the guestresidue of an EBP sequence and changing the composition ratio of theguest residue (see PROTEIN-PROTEIN INTERACTIONS: A MOLECULAR CLONINGMANUAL, 2002, Cold Spring Harbor Laboratory Press, Chapter 18. pp.329-343).

As described above, an EBP exhibits hydrophilicity when the EBP has ahigh T_(t), and hydrophobicity when the EBP has a low T_(t). Similarly,in the case of the EBP block according to the present invention, it isalso possible to increase or decrease T_(t) by changing an amino acidsequence including guest residues and a molecular weight thereof. Thus,a hydrophilic or hydrophobic EBP block may be prepared.

For reference, an EBP having T_(t) lower than a body temperature may beused as a hydrophobic block, whereas an EBP having T_(t) higher than abody temperature may be used as a hydrophilic block. Due to thisproperty of EBPs, the hydrophilic and hydrophobic properties of EBPs maybe relatively defined when EBPs are applied to biotechnology.

Taking EBP sequences according to the present invention as an example,when a plastic polypeptide block in which a plastic pentapeptide ofVal-Pro-Ala-X_(aa)-Gly is repeated is compared with an elasticpolypeptide block in which an elastic pentapeptide ofVal-Pro-Gly-X_(aa)-Gly is repeated, the third amino acid, Gly, hashigher hydrophilicity than Ala. Accordingly, the plastic polypeptideblock (elastin-based polypeptide with plasticity: EBPP) exhibits a lowerT_(t) than the elastic polypeptide block (elastin-based polypeptide withelasticity: EBPE).

EBPs according to the present invention, as described above, may exhibithydrophilic or hydrophobic properties by adjusting T_(t) and may becharged using charged amino acids.

Fusion polypeptides according to the present invention is schematicallyshown in FIGS. 5 b and 5 c . According to the present invention, an EBPwas fused to an anti-Flt1 peptide that specifically binds to VEGFreceptors (VEGFRs). Using the fusion polypeptide of the presentinvention, VEGF does not bind to VEGFRs, and thus neovascularization maybe inhibited.

The term “EBP diblock” used herein refers to a block composed of“hydrophilic EBP-hydrophobic EBP”, and is also called “EBP diblockcopolymer”, “EBP diblock block”, “diblock EBP” “diblock EBPs” or“diblock EBPPs”.

The present invention relates to a new class of genetically encoded“stimuli-responsive VEGFR-targeting fusion polypeptides”.

In one embodiment, specifically, the fusion polypeptide is composed ofan anti-Flt1 peptide acting as a receptor antagonist targeting VEGFR1and a hydrophilic EBP block as a soluble unimer.

In another embodiment, the fusion polypeptide is composed of ananti-Flt1 peptide acting as a receptor antagonist targeting VEGFR1; ahydrophilic EBP block; and a hydrophobic EBP block. The EBP diblock ofthe hydrophilic EBP block and the hydrophobic EBP block contributes tothe formation of a temperature-triggered core-shell micellar structure.

When using a fusion polypeptide composed of an anti-Flt1 peptide and ahydrophilic EBP block, a strong non-covalent interaction between VEGFR1and the anti-Flt1 peptide domain of the fusion polypeptide occurs.

In addition, a fusion polypeptide composed of an anti-Flt1 peptide; ahydrophilic EBP block; and a hydrophobic EBP block may form atemperature-triggered core-shell micellar structure with a multivalentanti-Flt1 peptide under physiological conditions, which may increase thebinding affinity of the fusion polypeptide for VEGFR1.

Therefore, the fusion polypeptide of the present invention may bepresented as a polypeptide drug for treating neovascularization-relateddiseases.

The fusion polypeptide of the present invention may overcome thefollowing limitations that arise when conventional peptide drugs andpeptide-polymer conjugates are applied to in vivo treatment: (1) rapidclearance by proteases; (2) time consuming and costly conjugation andpurification; (3) random distribution and (appearing upon polymerizationof various polymers and peptide drugs) inconsistent conjugationefficiency; and (4) heterogeneous micellar structures due topolydisperse molecular weights thereof.

The present invention provides a VEGFR-targeting fusion polypeptidecomposed of thermally responsive elastin-based polypeptides (EBPs) and avascular endothelial growth factor receptor (VEGFR)-targeting peptide.The fusion polypeptide of the present invention was geneticallyengineered, expressed and purified, and the physicochemical propertiesthereof were analyzed. The EBPs were introduced as non-chromatographicpurification tags and were also introduced as a stabilizer, like apoly(ethylene glycol) conjugate, for minimizing rapid in vivodegradation of VEGFR1-targeting peptides. In addition, theVEGFR-targeting peptide was introduced to function as a receptorantagonist by specifically binding to VEGFRs. A fusion polypeptidecomposed of a VEGFR-targeting peptide-hydrophilic EBP block exhibited asoluble unimer form. On the other hand, a fusion polypeptide composed ofVEGFR-targeting peptide-hydrophilic EBP block-hydrophobic EBP blockexhibited a temperature-triggered core-shell micellar structure with amultivalent VGFR-targeting peptide under physiological conditions. Asanalyzed by enzyme-linked immunosorbent assay (ELISA), these structuresincreased the binding affinity of a fusion polypeptide for VEGFreceptors (see Examples below). Depending on the spatial display of aVEGFR-targeting peptide, the binding affinity of the VEGFR-targetingpeptide to VEGFRs was greatly regulated. The present invention shows howthe binding affinity of a VEGFR-targeting peptide can be regulated basedon multivalency.

A therapeutic composition including a fusion polypeptide for inhibitingneovascularization according to the present invention is apharmaceutical composition. The pharmaceutical composition may includethe fusion polypeptide and other materials that do not interfere withthe function of the composition used in vivo for inhibitingneovascularization. Such other materials are not limited and may includediluents, excipients, carriers and/or other inhibitors ofneovascularization.

In some embodiments, the fusion polypeptides for inhibitingneovascularization of the present invention are formulated forconventional human administration, for example, by formulating thefusion polypeptides with a suitable diluent, including sterile water andnormal saline.

Administration or delivery of a therapeutic composition according to thepresent invention may be performed through any route so long as targettissues can be reached through that route. For example, theadministration may be performed by direct injection into a target tissue(e.g., cardiac tissue) such as topical or intradermal, subcutaneous,intramuscular, intraperitoneal, intraarterial, intracoronary,intrathymic or intravenous injection, or intravitreal injection. Thestability and/or potency of the fusion polypeptide disclosed in thepresent invention allow for convenient administration routes includingsubcutaneous, intradermal, intravenous and intramuscular routes.

The present invention provides a method of delivering a fusionpolypeptide (e.g., as a part of a composition or formulation describedherein) into cells, and a method of treating, alleviating, or preventingprogression of a disease in a subject. The term “subject” or “patient”used in the present invention refers to any vertebrate animals,including, without being limited to, humans and other primates (e.g.,chimpanzees and other apes and monkey species), farm animals (e.g.,cows, sheep, pigs, goats and horses), domestic mammals (e.g., dogs andcats), laboratory animals (e.g., rodents such as mice, rats and guineapigs) and birds (e.g., domestic, wild and game birds such as chickens,turkeys and poultry, ducks, geese, and the like). In some embodiments,the subject is a mammal.

In another embodiment, the mammal is a human.

A fusion polypeptide or pharmaceutical composition of the presentinvention may contact a target cell (e.g., a mammalian cell) in vitro orin vivo.

According to yet another aspect of the present invention, there isprovided a method of treating or preventing diseases caused byneovascularization, the method including a step of administering atherapeutic composition according to the present invention toindividuals.

For clinical use, the fusion polypeptide of the present invention may beadministered alone via any suitable route of administration effective toachieve a desired therapeutic result or may be formulated into apharmaceutical composition. The administration “route” of theoligonucleotides of the present invention may include enteral,parenteral and topical administration or inhalation. Among theadministration routes of the fusion polypeptide of the presentinvention, enteral includes oral, gastrointestinal, intestinal, andrectal. Parenteral routes include ocular injection, intravenous,intraperitoneal, intramuscular, intraspinal, subcutaneous, topical,vaginal, topical, nasal, mucosal and pulmonary administration. Thetopical route of administration of the fusion polypeptides of thepresent invention refers to external application of the oligonucleotidesinto the epidermis, mouth and ears, eyes and nose.

The therapeutic composition may be administered by parenteral, oral,transdermal, sustained release, controlled release, delayed release,suppository, catheter or sublingual administration.

When the fusion polypeptide included in the therapeutic composition isadministered in combination with other drugs, the fusion polypeptide maybe administered in an amount of 15 μg/kg or less when injectedintravenously, and may be administered in an amount of 2.5 μg or lesswhen injected intravitreally.

The present invention is further illustrated by the following additionalexamples which should not be construed as limiting. It should beunderstood by those of ordinary skill in the art that various changes tothe specific embodiments disclosed may be made without departing fromthe spirit and scope of the invention in the light of the presentinvention and that equivalent or similar results may be obtained.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by those of ordinary skillin the art to which this invention belongs. In general, the nomenclatureused herein is well known and commonly used in the art.

BRIEF DESCRIPTION OF THE DRAWINGS

The above and other objects, features and advantages of the presentinvention will become more apparent to those of ordinary skill in theart by describing in detail exemplary embodiments thereof with referenceto the accompanying drawings, in which:

FIG. 1 illustrates a schematic diagram and an adapter sequence forconstruction of plasmids encoding EBP gene libraries with different DNAsizes. (A) an adapter sequence for modification of a pET-21a plasmid,(B) a scheme for modification of a pET-21a plasmid for seamless genecloning, (C) a scheme for inserting a monomer EBP gene into a modifiedpET-21a vector, and (D) a scheme for construction of plasmids encodingEBP gene libraries with different DNA sizes;

FIG. 2 shows the agarose gel electrophoresis images of EBP genelibraries used in the present invention. (A) EBPE[A₁G₄I₁], (B)EBPP[A₁G₄I₁], (C) EBPE[K₁G₄I₁], (D) EBPP[K₁G₄I₁], (E) EBPE[D₁G₄I₁], (F)EBPP[D₁G₄I₁], (G) EBPE[E₁G₄I₁], (H) EBPP[E₁G₄I₁], (I) EBPP[G₁A₃F₂], (J)EBPP[K₁A₃F₂], (K) EBPP[D₁A₃F₂], and (L) EBPP[H₃A₂I₁]. The number of EBPrepeat units was indicated below each DNA band. Two side-lanes on allagarose gels represent different DNA size markers (0.1, 0.2, 0.3, 0.4,0.5, 0.6, 0.7, 1.0, 1.5, 2.0, and 3.0 kbp, from bottom to top);

FIG. 3 shows the copper-stained SDS-PAGE gel (4 to 20% gradient) imagesof EBPs used in the present invention. (A) EBPE[A₁G₄I₁], (B)EBPP[A₁G₄I₁], (C) EBPE[K₁G₄I₁], (D) EBPP[K₁G₄I₁], (E) EBPE[D₁G₄I₁] and(F) EBPP[D₁G₄I₁]. Two side-lanes on SDS-PAGE gels contain standardprotein size markers (7, 15, 24, 35, 40, 50, 65, 90, 110, and 150 kDa,from bottom to top);

FIGS. 4A to 4F show the thermal profiles of EBPs used in the presentinvention. FIG. 4A shows the profiles of EBPE[A₁G₄I₁]_(n), FIG. 4B showsthe profiles of EBPP[A₁G₄I₁]_(n), FIG. 4C shows the profiles ofEBPE[K₁G₄I₁]_(n), FIG. 4D shows the profiles of EBPP[K₁G₄I₁]_(n), FIG.4E shows the profiles of EBPP[D₁G₄I₁]_(n), and FIG. 4F shows theprofiles of EBPP[G₁A₃F₂]_(n). To obtain thermal profiles, 25 μM EBPsolutions were prepared in PBS buffer or PBS buffer supplemented with 1to 3 M sodium chloride, and the optical absorbance of the EBP solutionwas measured at 350 nm while heating the solution at a heating rate of1° C./min;

FIGS. 5A to 5D are schematic diagrams of cloning, molecular structures,and functions of fusion polypeptides composed of EBPPs and an anti-Flt1peptide. In FIG. 5A genes encoding EBPP diblocks were constructed, and agene encoding an anti-Flt1 peptide was cloned into a plasmid including agene encoding an EBPP diblock. In FIG. 5B fusion polypeptides composedof anti-Flt1 peptide-hydrophilic EBP; and anti-Flt1 peptide-hydrophilicEBP-hydrophobic EBP. In FIG. 5C fusion polypeptides composed ofanti-Flt1 peptide-hydrophilic EBP-hydrophobic EBP were able to form amicellar structure by a temperature stimulus. In FIG. 5D (i) fusionpolypeptides composed of anti-Flt1 peptide-hydrophilic EBP were able tobind to VEGFRs, and were able to inhibit interactions between VEGFR1 andVEGF. (ii) The micellar structures of fusion polypeptides composed ofanti-Flt1 peptide-hydrophilic EBP-hydrophobic EBP were able to bind toVEGFRs, and was able to inhibit interactions between VEGFRs and VEGFwith increased affinity due to the multivalency of the anti-Flt1peptide;

FIG. 6 shows (A) agarose gel (1%) images and (B) SDS-PAGE (4 to 20%gradient) gel images. (a) anti-Flt1-EBPP[A₁G₄I₁]₃, (b)anti-Flt1-EBPP[A₁G₄I₁]₆, (c) anti-Flt1-EBPP[A₁G₄I₁]₁₂ and (d)anti-Flt1-EBPP[A₁G₄I₁]₂₄;

FIG. 7 shows the LCST of EBPP[A₁G₄I₁]_(3n) (n: integer) andanti-Flt1-EBPP[A₁G₄I₁]_(3n) (n: integer) as turbidity profiles.Turbidity profiles were determined by measuring the absorbance of (A toC) 25 μM EBPP[A₁G₄I₁]_(3n) (n: integer) and (D to F) 25 μManti-Flt1-EBPP[A₁G₄I₁]_(3n) (n: integer). The absorbance was measured at350 nm in 10 mM PBS (A and D), 10 mM PBS supplemented with 1 M sodiumchloride (B and E), and 10 mM PBS supplemented with 2 M sodium chloride(C and F), while heating samples at a heating rate of 1° C./min;

FIG. 8 shows the (A) agarose gel (1%) images and the (B) SDS-PAGE (4 to20% gradient) gel images of fusion polypeptides. (A) A modified pET21-a(+) plasmid encoding anti-Flt1-EBPP[E₁G₄I₁]₁₂-[G₁A₃F₂]₁₂ oranti-Flt1-EBPP[E₁G₄I₁]₁₂-[G₁A₃F₂]₂₄ was digested by XbaI and BseRI. (B)The fusion polypeptides were expressed in E. coli and purified by ITC. 4to 20% gradient gels were visualized with copper stain. An expectedmolecular weight was indicated below the band;

FIG. 9 shows the turbidity profiles of EBPP diblocks depending on thepresence or absence of an anti-Flt1 peptide. (A) The turbidity profilesof (a) 25 μM EBPP[E₁G₄I₁]₁₂-EBPP[G₁A₃F₂]₁₂ and (b) 25 μMEBPP[E₁G₄I₁]₁₂-EBPP[G₁A₃F₂]₂₄. (B) The turbidity profiles ofanti-Flt1-EBPP[E₁G₄I₁]₁₂-EBPP[G₁A₃F₂]₁₂ and (C) the turbidity profilesof anti-Flt1-EBPP[E₁G₄I₁]₁₂-EBPP[G₁A₃F₂]₂₄ were obtained forconcentrations of 12.5, 25, 50 and 100 μM in 10 mM PBS. Absorbance wasmeasured at 350 nm while heating the samples at a rate of 1° C./min. Aphase transition occurred twice. The first phase transition occurred asa result of hydrophobic block aggregation, the second phase transitionwas affected by polar EBPP[E₁G₄I₁]₁₂. As EBPP diblock concentrationincreased, the first and second T_(t) values thereof were lowered;

FIG. 10 shows the hydrodynamic radius of (a)anti-Flt1-EBPP[E₁G₄I₁]₁₂-EBPP[G₁A₃F₂]₁₂ and (b)anti-Flt1-EBPP[E₁G₄I₁]₁₂-EBPP[G₁A₃F₂]₂₄, and the hydrodynamic radius wasmeasured by a DLS instrument. The hydrodynamic radius of EBPP diblockpolypeptides was measured at 25 μM in 10 mM PBS. The hydrodynamic radiusof EBPP diblock polypeptides prior to the first phase transition is lessthan 10 nm, indicating that the polypeptides are present in a solubleunimer form;

FIG. 11 shows the in vitro biological activities of EBPP[A₁G₄I₁]₁₂,anti-Flt1-EBPP[A₁G₄I₁]₃, anti-Flt1-EBPP[A₁G₄I₁]₆,anti-Flt1-EBPP[A₁G₄I₁]₁₂ and anti-Flt1-EBPP[A₁G₄I₁]₂₄, which inhibitVEGFR1 binding to coated VEGF;

FIG. 12 shows the in vitro biological activities of EBPP[A₁G₄I₁]₁₂,anti-Flt1-EBPP[A₁G₄I₁]₁₂, anti-Flt1-EBPP[E₁G₄I₁]₁₂-EBPP[G₁A₃F₂]₁₂ andanti-Flt1-EBPP[E₁G₄I₁]₁₂-EBPP[G₁A₃F₂]₂₄, which inhibit VEGFR binding tocoated VEGF. EBPP[A₁G₄I₁]₁₂ and anti-Flt1-EBPP[A₁G₄I₁]₁₂ were unimers at37° C. On the other hand, anti-Flt1-EBPP[E₁G₄I₁]₁₂-EBPP[G₁A₃F₂]₁₂ formedmetastable micelles at 37° C., andanti-Flt1-EBPP[E₁G₄I₁]₁₂-EBPP[G₁A₃F₂]₂₄ formed stable micelles;

FIG. 13 shows the results of the in vitro tube formation assay ofanti-Flt1-EBPP[A₁G₄I₁]₁₂. (A) the fluorescence microscope images ofcalcein-AM-labeled HUVECs and (B) the degree of inhibition of tubeformation. The degree of inhibition of tube formation was quantifiedfrom the images of (A). The tube length of HUVECs treated withanti-Flt1-EBPP[A₁G₄I₁]₁₂ decreased with increasing the concentration ofanti-Flt1-EBPP[A₁G₄I₁]. The anti-Flt1-EBPP [A₁G₄I₁]₁₂ inhibitedmigration and tube formation of HUVECs. Tubing lengths are averagevalues±SE. *P≤0.05 by a t test; and

FIG. 14 shows an in vivo inhibition effect of anti-Flt1-EBPP[A₁G₄I₁]₁₂in a laser-induced choroidal neovascularization model. C57BL6 mice (n=3per group) were treated with a vehicle (PBS), EBPP[A₁G₄I₁]₁₂ (20 μg) oranti-Flt1-EBPP[A₁G₄I₁]₁₂ (0.1, 1, 5 and 20 μg) after laser-inducedinjury, and the treatment was continued for 5 days. At day 14 afterlaser injury, mice were euthanized and fluorescein isothiocyanate(FITC)-dextran perfused whole choroidal flat-mounts were prepared. TheCNV lesion size was quantified by Nano-Zoomer and FISH. (A)representative flat mount fluorescence microscopic images. (B) a graphof the CNV size of each treated group. Each point corresponds to a CNVlesion, and a horizontal bar corresponds to the average value of eachgroup. *P≤0.05 by an unpaired t test. The data represents twoindependent experiments.

DETAILED DESCRIPTION OF EXEMPLARY EMBODIMENTS Example 1: Materials

A pET-21a (+) vector and BL21 (DE3) E. Coli cells were obtained fromNovagen Inc. (Madison, Wis., U.S.). Top10 competent cells and calcein-AMwere purchased from Invitrogen (Carlsbad, Calif., U.S.) and HUVECs werepurchased from American Type Culture Collection (ATCC) (Virginia, U.S.).All customized oligonucleotides were synthesized by Cosmo GeneTech(Seoul, South Korea) and recombinant human VEGF-165 (rhVEGF₁₆₅) wasobtained from Sino Biological Inc. (Beijing, China). Calf intestinalalkaline phosphatase (CIP), BamHI and XbaI were obtained from Fermentas(Ontario, Canada). AcuI and BseRI were purchased from New EnglandBiolabs (Ipswich, Mass., U.S.). T4 DNA ligase was obtained from ElpisBio-tech (Taejeon, South Korea). DNA miniprep, gel extraction, and PCRpurification kits were obtained from Geneall Biotechnology (Seoul, SouthKorea). “Dyne Agarose High” was obtained from DYNE BIO, Inc. (Seongnam,South Korea). Top10 cells were grown in “TB DRY” media obtained from MOBIO Laboratories, Inc. (Carlsbad. Calif., U.S.). BL21 (DE3) cells weregrown in “CircleGrow” media obtained from MP Biomedicals (Solon, Ohio,U.S.). “Ready Gels, Tris-HCl 2-20% precast gels” were from Bio-Rad(Hercules, Calif., U.S.). Phosphate buffered saline (PBS, pH 7.4),kanamycin, polyethyleneamine (PEI), FITC-dextran, formalin and bovineserum albumin (BSA) were obtained from Sigma-Aldrich (St Louis, Mo.,U.S.). Matrigel was purchased from BD Biosciences (San Diego, Calif.,U.S.). Avastin, also known as bevacizumab was purchased from RochePharma Ltd. (Reinach, Switzerland). Ketamine was obtained from Huons(Seongnam, South Korea). Xylazine was purchased from BAYER (Leverkusen,Germany). Tropicamide was purchased from Santen Pharmaceutical Co. Ltd(Kita-ku, Osaka, Japan). A stereomicroscope was obtained from Leica(Wetzlar, Germany). Recombinant human VEGF₁₆₅ protein and recombinanthuman VEGF R1/Flt-1 F_(c) were purchased from R&D System (Minneapolis,Minn., U.S.). Rabbit anti-human IgG F_(c)-HRP chimeric protein and3,3′,5,5′-tetramethylbenzidine (TMB) was obtained from ThermoFisher(Massachusetts, U.S.).

Example 2: Notation for Different EBP Blocks and Block PolypeptidesThereof

Different EBPs having a pentapeptide repeat unit of Val-Pro-(Gly orAla)-X_(aa)-Gly[VP (G or A)XG] are named as follows. X_(aa) may be anyamino acid except Pro. First, pentapeptide repeats ofVal-Pro-Ala-X_(aa)-Gly (VPAXG) with plasticity are defined as anelastin-based polypeptide with plasticity (EBPP). On the other hand,pentapeptide repeats of Val-Pro-Gly-X_(aa)-Gly (VPGXG) are calledelastin-based polypeptides with elasticity (EBPEs). Second, in[X_(i)Y_(j)Z_(k)]_(n), the capital letters in the parentheses representthe single letter amino acid codes of guest residues, i.e., amino acidsat the fourth position (X_(aa) or X) of an EBP pentapeptide, andsubscripts corresponding to the capital letters indicate the ratio ofthe guest residues in an EBP monomer gene as a repeat unit. Thesubscript number n of [X_(i)Y_(j)Z_(k)]_(n) represents the total numberof repeats of an EBP corresponding to SEQ ID NO. 1 [VPGXG VPGXG VPGXGVPGXG VPGXG VPGXG] or SEQ ID NO. 2[VPAXG VPAXG VPAXG VPAXG VPAXG VPAXG]according to the present invention. For example, EBPP[G₁A₃F₂]₁₂ is anEBPP block including 12 repeats of a pentapeptide unit, SEQ ID NO. 2[VPAXG VPAXG VPAXG VPAXG VPAXG VPAXG], in which a ratio of Gly, Ala, andPhe at the fourth guest residue position (X_(aa)) is 1:3:2. Finally,EBP-EBP diblock polypeptides are named according to the composition ofeach block in brackets with a hyphen between blocks as inEBPP[E₁G₄I₁]₁₂-EBPP[G₁A₃F₂]₁₂.

Example 3: Preparation of Modified pET-21a Vector for Cloning SeamlessGene

4 μg of a pET-21a vector was digested and dephosphorylated with 50 U ofXbaI, 50 U of BamHI and 10 U of a thermosensitive alkaline phosphatasein FastDigest buffer for 20 minutes at 37° C. The digested plasmid DNAwas purified using a PCR purification kit, and then was eluted in 40 μlof distilled and deionized water. Two oligonucleotides with XbaI andBamHI compatible sticky ends were designed, i.e., SEQ ID NO. 39(5′-ctagaaataattttgtttaactttaagaaggaggagtacatatgggctactgataatgatcttcag-3′)and SEQ ID NO. 40(5′-gatcctgaagatcattatcagtagcccatatgtactcctccttcttaaagttaaacaaaattattt-3′).To anneal the two types of oligonucleotides, each oligonucleotide wasprepared at a concentration of 2 μM in 50 μl of T4 DNA ligase buffer,heat treated at 95° C. for 2 minutes and then slowly cooled to roomtemperature over 3 hours. To ligate the annealed dsDNA, i.e., a DNAinsert, into multiple cloning sites within the linearized pET-21avector, 20 pmol of the annealed dsDNA and 0.1 pmol of the linearizedpET-21a vector were incubated in T4 DNA ligase buffer containing 1 U ofT4 DNA ligase for 30 minutes at 37° C. The modified pET-21a (mpET-21a)vector for cloning and expressing a seamless gene was transformed intoTop10 competent cells, followed by plating the Top10 competent cells ona super optimal broth with catabolite repression (SOC) platesupplemented with 50 μg/ml ampicillin. The DNA sequence of the mpET-21avector was then verified by fluorescent dye terminator DNA sequencing(Applied Biosystems Automatic DNA Sequencer ABI3730).

Example 4: Synthesis of EBP Monomer Gene and Oligomerization Thereof

EBP sequences having a pentapeptide repeat unit, Val-Pro-(Gly orAla)-X_(aa)-Gly, in which the fourth residues were varied in differentmolar ratios, were designed at the DNA level to optimize T_(t) below aphysiological temperature. The DNA and amino acid sequences of EBPs withvarious pentapeptide repeat units for 17 EBP libraries are shown inTables 1 and 2, respectively.

TABLE 1 Gene sequences corresponding to EBP libraries. Both EBPs withplasticity (EBPPs) having a pentapeptide repeat of Val-Pro-Ala-X_(aa)-Gly, and EBPs with elasticity (EBPEs)having a pentapeptide repeat of Val-Pro-Gly-X_(aa)-Glywere cloned to have the same guest residue composition and ratio. SEQ IDEBP Gene Sequence NO. EBPE[A₁G₄I₁]GTC CCA GGT GGA GGT GTA CCC GGC GCG GGT GTC CCA GGT GGA GGT 3GTA CCT GGG GGT GGG GTC CCT GGT ATT GGC GTA CCT GGA GGC GGC EBPP[A₁G₄I₁]GTT CCA GCT GGC GGT GTA CCT GCT GCT GCT GTT CCG GCC GGT GGT 4GTT CCG GCG GGC GGC GTG CCT GCA ATA GGA GTT CCC GCT GGT GGC EBPE[K₁G₄I₁]GTT CCG GGT GGT GGT GTT CCG GGT AAA GGT GTT CCG GGT GGT GGT 5GTT CCG GGT GGT GGT GGT GTT CCG GGT ATC GGT GTT CCG GGT GGC EBPP[K₁G₄I₁]GTT CCG GCG GGT GGT GTT CCG GCG AAA GGT GTT CCG GCG GGT GGT 6GTT CCG GCG GGT GGT GTT CCG GCG ATC GGT GTT CCG GCG GGT GGC EBPE[D₁G₄I₁]GTT CCG GGT GGT GGT GTT CCG GGT GAT GGT GTT CCG GGT GGT GGT 7GTT CCG GGT GGT GGT GGT GTT CCG GGT ATC GGT GTT CCG GGT GGC EBPP[D₁G₄I₁]GTT CCG GCG GGT GGT GTT CCG GCG GAT GGT GTT CCG GCG GGT GGT 8GTT CCG GCG GGT GGT GTT CCG GCG ATC GGT GTT CCG GCG GGT GGC EBPE[E₁G₄I₁]GTT CCG GGT GGT GGT GTT CCG GGT GAA GGT GTT CCG GGT GGT GGT 9GTT CCG GGT GGT GGT GGT GTT CCG GGT ATC GGT GTT CCG GGT GGC EBPP[E₁G₄I₁]GTT CCG GCG GGT GGT GTT CCG GCG GAA GGT GTT CCG GCG GGT GGT 10GTT CCG GCG GGT GGT GTT CCG GCG ATC GGT GTT CCG GCG GGT GGC EBPE[G₁A₃F₂]GTC CCG GGT GCG GGC GTG CCG GGA TTT GGA GTT CCG GGT GCG GGT 11GTT CCA GGC GGT GGT GTT CCG GGC GCG GGC GTG CCG GGC TTT GGC EBPP[G₁A₃F₂]GTG CCG GCG GCG GGC GTT CCA GCC TTT GGT GTG CCA GCG GCG GGA 12GTT CCG GCC GGT GGC GTG CCG GCA GCG GGC GTG CCG GCT TTT GGC EBPP[K₁A₃F₂]GTG CCG GCG GCG GGC GTT CCA GCC TTT GGT GTG CCA GCG GCG GGA 13GTT CCG GCC AAA GGC GTG CCG GCA GCG GGC GTG CCG GCT TTT GGC EBPP[D₁A₃F₂]GTG CCG GCG GCG GGC GTT CCA GCC TTT GGT GTG CCA GCG GCG GGA 14GTT CCG GCC GAT GGC GTG CCG GCA GCG GGC GTG CCG GCT TTT GGC EBPP[K₃F₃]GTT CCA GCG TTT GGC GTG CCA GCG AAA GGT GTT CCG GCG TTT GGG 15GTT CCC GCG AAA GGT GTG CCG GCC TTT GGT GTG CCG GCC AAA GGC EBPP[D₃F₃]GTT CCA GCG TTT GGC GTG CCA GCG GAT GGT GTT CCG GCG TTT GGG 16GTT CCC GCG GAT GGT GTG CCG GCC TTT GGT GTG CCG GCC GAT GGC EBPP[H₃A₃I₁]GTG CCG GCG CAT GGA GTT CCT GCC GCC GGT GTT CCT GCG CAT GGT 17GTA CCG GCA ATT GGC GTT CCG GCA CAT GGT GTG CCG GCC GCC GGC EBPP[H₅G₁]GTT CCG GCC GGA GGT GTA CCG GCG CAT GGT GTT CCG GCA CAT GGT 18GTG CCG GCT CAC GGT GTG CCT GCG CAT GGC GTT CCT GCG CAT GGC EBPP[G₁C₃F₂]GTG CCG GCG TGC GGC GTT CCA GCC TTT GGT GTG CCA GCG TGC GGA 19GTT CCG GCC GGT GGC GTG CCG GCA TGC GGC GTG CCG GCT TTT GGC

TABLE 2 Amino acid sequences corresponding to EBP libraries SEQ ID EBPAmino acid Sequence NO. EBPE[A₁G₄I₁] VPGGG VPGAG VPGGG VPGGG VPGIG VPGGG20 EBPP[A₁G₄I₁] VPAGG VPAAG VPAGG VPAGG VPAIG VPAGG 21 EBPE[K₁G₄I₁]VPGGG VPGKG VPGGG VPGGG VPGIG VPGGG 22 EBPP[K₁G₄I₁]VPAGG VPAKG VPAGG VPAGG VPAIG VPAGG 23 EBPE[D₁G₄I₁]VPGGG VPGDG VPGGG VPGGG VPGIG VPGGG 24 EBPP[D₁G₄I₁]VPAGG VPADG VPAGG VPAGG VPAIG VPAGG 25 EBPE[E₁G₄I₁]VPGGG VPGEG VPGGG VPGGG VPGIG VPGGG 26 EBPP[E₁G₄I₁]VPAGG VPAEG VPAGG VPAGG VPAIG VPAGG 27 EBPE[G₁A₃F₂]VPGAG VPGFG VPGAG VPGGG VPGAG VPGFG 28 EBPP[G₁A₃F₂]VPAAG VPAFG VPAAG VPAGG VPAAG VPAFG 29 EBPP[K₁A₃F₂]VPAAG VPAFG VPAAG VPAGG VPAAG VPAFG 30 EBPP[D₁A₃F₂]VPAAG VPAFG VPAAG VPAGG VPAAG VPAFG 31 EBPP[K₃F₃]VPAFG VPAKG VPAFG VPAKG VPAFG VPAKG 32 EBPP[D₃F₃]VPAFG VPADG VPAFG VPADG VPAFG VPADG 33 EBPP[H₃A₃I₁]VPAHG VPAAG VPAHG VPAIG VPAHG VPAAG 34 EBPP[H₅G₁]VPAGG VPAHG VPAHG VPAHG VPAHG VPAHG 35 EBPP[G₁C₃F₂]VPACG VPAFG VPACG VPAGG VPACG VPAFG 36

In Table 1, SEQ ID NO. 3 to 10 may be classified as gene sequences forhydrophilic EBP blocks, and SEQ ID NO. 11 to 19 may be classified asgene sequences for hydrophobic EBP blocks, in which Phe and His areincorporated. In Table 2, amino acid SEQ ID NO. 20 to 27 may beclassified as hydrophilic EBP blocks, and amino acid SEQ ID NO. 28 to36, in which Phe and His are incorporated, may be classified ashydrophobic EBP blocks. In particular, in Table 2, SEQ ID NO. 22 and 23are classified as positively charged hydrophilic EBP blocks, and SEQ IDNO. 24 to 27 are classified as negatively charged hydrophilic EBPblocks. That is, as described above, when the LCST of an EBP is lowerthan the body temperature, the EBP exhibits hydrophobicity, and when theLCST of an EBP is higher than the body temperature, the EBP exhibitshydrophilicity. Due to this nature of EBPs, the hydrophilic andhydrophobic properties of EBPs may be relatively defined when EBPs areapplied to biotechnology.

Different EBPs having a pentapeptide repeat unit, Val-Pro-(Gly orAla)-X_(aa)-Gly [where X_(aa) may be any amino acid except Pro], whichare capable of responding to unique stimuli including temperature andpH, were designed at the DNA level. EBPs with plasticity (EBPPs) havinga pentapeptide repeat unit of Val-Pro-Ala-X_(aa)-Gly and EBPs withelasticity (EBPEs) having a pentapeptide repeat unit ofVal-Pro-Gly-X_(aa)-Gly were all cloned to have the same guest residuecomposition and ratio. Tables 1 and 2 represent the gene and amino acidsequences of different EBPs having respective pentapeptide units. Forexample, EBPE[G₁A₃F₂]₁₂ and EBPP[G₁A₃F₂]₁₂ not only show almost the samemolar mass, but also the fourth residues of these EBP pentapeptide unitsrepresent the same combination. In addition, these EBP blocks havedifferent mechanical properties because the third amino acid residues(Ala or Gly) of the pentapeptide units are different. Positively andnegatively charged EBPs were prepared by introducing charged amino acidssuch as Lys, Asp, GIu, and His as guest residues.

To anneal each pair of oligonucleotides encoding various EBPs, eacholigonucleotide was prepared at a concentration of 2 μM in 50 μl of T4DNA ligase buffer, heat treated at 95° C. for 2 minutes and then slowlycooled to room temperature over 3 hours. 4 μg of a modified pET-21avector was digested and dephosphorylated with 15 U of BseRI and 10 U ofFastAP thermosensitive alkaline phosphatase for 30 minutes at 37° C. Thedigested plasmid DNA was purified using a PCR purification kit, and thenwas eluted in 40 μl of distilled and deionized water. To ligate theannealed dsDNA, i.e., a DNA insert, into multiple cloning sites withinthe linearized mpET-21a vector, 90 pmol of the annealed dsDNA and 30pmol of the linearized mpET-21a vector were incubated in T4 DNA ligasebuffer containing 1 U of T4 DNA ligase for 30 minutes at 16° C. Theligated plasmid was transformed into Top10 chemically competent cells,followed by plating the Top10 competent cells on an SOC platesupplemented with 50 μg/ml ampicillin. DNA sequences were then confirmedby DNA sequencing. After all EBP monomer genes were constructed, eachEBP gene was synthesized by ligating each of 36 types of repetitivegenes (as an insert) into the corresponding vector containing each ofthe same 36 types of repetitive genes, as follows. A cloning procedurefor EBP libraries and fusions thereof are illustrated in FIG. 1 .Vectors harboring gene copies corresponding to EBP monomers weredigested and dephosphorylated with 10 U of XbaI, 15 U of BseRI and 10 Uof FastAP thermosensitive alkaline phosphatase in CutSmart buffer for 30minutes at 37° C. The digested plasmid DNA was purified using a PCRpurification kit, and then was eluted in 40 μl of distilled anddeionized water. For preparation of an insert part, a total of 4 μg ofan EBP monomer gene was digested with 10 U of XbaI and 15 U of AcuI inCutSmart buffer for 30 minutes at 37° C. After digestion, the reactionproduct was separated by agarose gel electrophoresis and the insert waspurified using a gel extraction kit. Ligation was performed byincubating 90 pmol of the purified insert with 30 pmol of the linearizedvector in T4 DNA ligase buffer containing 1 U of T4 DNA ligase for 30minutes at 16° C. The product was transformed into Top10 chemicallycompetent cells, and then the cells were plated on an SOC platesupplemented with 50 μg/ml ampicillin Transformants were initiallyscreened by diagnostic restriction digestion on an agarose gel andfurther confirmed by DNA sequencing as described above.

As described above, EBP gene libraries having different DNA sizes weresynthesized using the designed plasmid vector and three differentrestriction endonucleases. FIG. 1 illustrates a recursive directionalligation (RDL) method, in which EBP monomer genes are ligated to formoligomerized EBP genes. For example, a gene construct encodingEBPP[G₁A₃F₂]₁₂ was prepared by ligation, wherein a plasmid backbone andan insert derived from a plasmid-borne gene vector harboring a geneencoding EBPP[G₁A₃F₂]₆ were used. The plasmid-borne gene vectorharboring a gene encoding EBPP[G₁A₃F₂]₆ was double-digested by XbaI andAcuI to obtain an insert, i.e., a gene fragment encoding EBPP[G₁A₃F₂]₆.On the other hand, the plasmid-borne gene vector for EBPP[G₁A₃F₂]₆ wasdouble-digested by XbaI and BseRI to obtain a plasmid backbone and thenthe plasmid backbone was dephosphorylated by treatment with an alkalinephosphatase. The RDL method using two different double restrictionenzymes has several advantages. First, due to the different shapes ofthe protrusions of both an insert and a digested vector, self-ligationof the digested vector did not occur, and the insert and the digestedvector were efficiently linked in a head-tail orientation. Second, dueto the mechanism of type III restriction endonuclease, an additional DNAsequence encoding each linker between blocks is not required. Each EBPgene was oligomerized to generate 36, 72, 108, 144, 180, and 216 EBPpentapeptide repeats. Using two restriction endonucleases XbaI andBamHI, oligomerized genes with sizes of 540, 1080, 1620, 2160, 2700, and3240 base pairs (bps) were confirmed. As characterized by agarose gelelectrophoresis, FIG. 2 depicts the digested DNA bands of EBP librarieswith DNA size markers on both end lanes. For example, EBPE[A₁G₄I₁] inFIG. 2(A) clearly shows a digested DNA band corresponding to a DNAregion encoding an oligomerized pentapeptide sequence containing Ala,Gly, Ile in a ratio of 1:4:1 as a guest residue. All digested DNA bandsare shown as corresponding lengths as compared to the molecular sizemarkers.

EBP genes and block co-polypeptides thereof were overexpressed in E.coli having a T7 promoter and purified by multiple cycles of inversetransition cycling (ITC). FIG. 3 shows copper-stained SDS-PAGE gelimages of the purified EBPs. EBPs shifted at least 20% more thantheoretically calculated molecular weights. Two side-lanes on SDS-PAGEgels contain standard protein size markers (7, 15, 24, 35, 40, 50, 65,90, 110, and 150 kDa, from bottom to top). In FIGS. 3(A) and 3(B),EBPE[A₁G₄I₁] and EBPP[A₁G₄I₁] represent a series of correspondingproteins with a molecular weight greater than a theoretical molecularweight (for EBPE[A₁G₄I₁], 14.0, 27.7, 41.3, 55.0, and 68.6 kDa, fromleft to right). In general, as shown in FIGS. 3(C) and 3(D), positivelycharged EBP libraries, including EBPE[K₁G₄I₁] and EBPP[K₁G₄I₁], showedhigher molecular weights than nonpolar EBP libraries, includingEBPE[A₁G₄I₁] and EBPP[A₁G₄I₁]. In addition, as shown in FIGS. 3(E) and3(F), negatively charged EBP libraries, including EBPE[D₁G₄I₁] andEBPP[D₁G₁I₁], have differently charged characteristics, and thusexhibited higher molecular weights than positively charged EBPlibraries.

EBP libraries were characterized. FIGS. 4A to 4F show thermal transitionbehaviors of EBPs determined by measuring optical absorbance at 350 nm(absorbance₃₅₀) at a heating rate of 1° C./min Inverse transitiontemperature (T_(t)) is defined as a temperature at which the firstderivative (d (OD₃₅₀)/dT) of turbidity, which is a function oftemperature, was the maximum. Based on environmental conditions such asa salt concentration and pH and the different third and fourth aminoacids of an EBP pentapeptide repeat unit, the T_(t) of an EBP was finelycontrolled in PBS and PBS was supplemented with 1 to 3 M sodiumchloride. For example, EBPE[A₁G₄I₁]₁₂ (FIG. 4A) with Gly at the thirdamino acid of an EBP pentapeptide repeat exhibited a T_(t) about 15° C.higher than that of EBPP[A₁G₄I₁]₁₂ (FIG. 4B) with Ala at the third aminoacid of an EBP pentapeptide repeat in PBS containing 1 M sodiumchloride, because Gly at the third amino acid of an EBP pentapeptiderepeat has a higher hydrophilicity than Ala. In general, charged EBPlibraries have a higher T_(t) than nonpolar EBP libraries becausecharged residues are introduced into the fourth amino acid of the EBPpentapeptide repeat of the charged EBPs. Negatively charged EBPlibraries, such as EBPP[D₁G₄I₁] (FIG. 4E), have different pK_(a) valuesfor Asp and Lys at the fourth amino acid of an EBP pentapeptide repeat,and thus have a higher T_(t) than positively charged EBP libraries, suchas EBPE[K₁G₄I₁] (FIG. 4C) and EBPP[K₁G₄I₁] (FIG. 4D). For reference,FIGS. 4A, 4B, 4C, 4D and 4E exhibit hydrophilicity, and FIG. 4F exhibitsEBPP[G₁A₃F₂]₁₂ and EBPP[G₁A₃F₂]₂₄ exhibit hydrophobicity.

Example 5: Gene Construction of Anti-Flt1-EBPP[A₁G₄I₁]n andAnti-Flt1-EBP Diblock Block (Copolypeptides)

A pair of oligonucleotides encoding an anti-Flt1 peptide acting as aVEGFR1 antagonist were chemically synthesized by Cosmo Genetech (Seoul,Korea), and linked to an oligonucleotide cassette with cohesive endsincluding restriction sites recognized by AcuI and BseRI. Anoligonucleotide cassette encoding the anti-Flt1 peptide was rationallydesigned to have no restriction sites recognized by BseRI, XbaI, AcuIand BamHI for seamless gene cloning, as shown in Table 3.

TABLE 3 Gene and amino acid sequences of CPPs SEQ ID NO. Sequence TypeSequence 37 Gene Sequence GGC AAT CAG TGG TTT ATT 38 Amino acid G N Q W F I Sequence

In Table 4, the sequences, gene lengths and molecular weights of fusionpolypeptides with a hydrophilic EBP block or an EBP diblock ofhydrophilic EBP block-hydrophobic EBP block are shown.

TABLE 4 Sequences, gene lengths and molecular weightsof fusion polypeptides Nucleotide Fusion protein (SEQ ID NO.)length (bp) M.W (kDa) Anti-Flt1-EBPP[A₁G₄I₁]₃ (SEQ ID NO. 48) 288 8.19Anti-Flt1-EBPP[A₁G₄I₁]₆ (SEQ ID NO. 49) 558 15.27Anti-Flt1-EBPP[A₁G₄I₁]₁₂ (SEQ ID NO. 50) 1098 29.42Anti-Flt1-EBPP[A₁G₄I₁]₂₄ (SEQ ID NO. 51) 2178 57.72Anti-Flt1-EBPP[E₁G₄I₁]₁₂-EBPP[G₁A₃F₂]₁₂ 2178 59.90 (SEQ ID NO. 52)Anti-Flt1-EBPP[E₁G₄I₁]₁₂-EBPP[G₁A₃F₂]₂₄ 3258 90.00 (SEQ ID NO. 53)

Each plasmid containing an EBP with restriction sites recognized byBseRI, XbaI, AcuI and BamHI, and the oligonucleotide cassette were usedto create genes for the fusion polypeptide libraries ofanti-Flt1-EBPP[A₁G₄I₁]₃ and anti-Flt1-EBP diblock blocks. First, toanneal a pair of oligonucleotides encoding an anti-Flt1 peptide, eacholigonucleotide was prepared at a concentration of 2 μM in 50 μl of T4DNA ligase buffer, heat treated at 95° C. for 2 minutes and then thereaction solution was slowly cooled to room temperature over 3 hours. Toclone the anti-Flt1-EBPP[A₁G₄I₁]_(3n), a plasmid vector encodingEBPP[A₁G₄I₁]_(3n) was digested with 15 U of BseRI in CutSmart buffer for30 minutes at 37° C. The digested plasmid DNA was purified using a PCRpurification kit, and then dephosphorylated with 10 U of FastAP as athermosensitive alkaline phosphatase in CutSmart buffer for 1 hour at37° C. The digested and dephosphorylated plasmid DNA was purified usinga PCR purification kit, and then eluted in 40 μl of distilled anddeionized water. Ligation was performed by incubating 90 pmol of thepurified insert and 30 pmol of the linearized vector in T4 DNA ligasebuffer containing 1 U of T4 DNA ligase at 16° C. for 30 minutes. Theproduct was transformed into Top10 chemically competent cells and thecells were plated on SOC plates supplemented with 50 μg/ml ampicillinTransformants were initially screened by diagnostic restrictiondigestion on an agarose gel and further confirmed by DNA sequencing asdescribed above.

Similarly, to clone anti-Flt1-EBP diblock blocks with hydrophobic blocksof different lengths, plasmid vectors encoding EBPP[G₁A₃F₂]_(n) weredigested with 10 U of XbaI and 15 U of BseRI in CutSmart buffer for 30minutes at 37° C. The digested plasmid DNA was purified using a PCRpurification kit, and then dephosphorylated with 10 U of FastAP as athermosensitive alkaline phosphatase in CutSmart buffer for 1 hour at37° C. The digested and dephosphorylated plasmid DNA was purified usinga PCR purification kit, and then eluted in 40 μl of distilled anddeionized water. 4 μg of EBPP[E₁G₄I₁]_(n) genes were digested with 10 Uof XbaI and 15 U of AcuI in CutSmart buffer for 30 minutes at 37° C.After digestion, the reaction product was separated by agarose gelelectrophoresis and an insert was purified using a gel extraction kit.Ligation was performed by incubating 90 pmol of the purified insert and30 pmol of the linearized vector in T4 DNA ligase buffer containing 1 Uof T4 DNA ligase at 16° C. for 30 minutes. The product was transformedinto Top10 chemically competent cells and the cells were plated on SOCplates supplemented with 50 μg/ml ampicillin Transformants wereinitially screened by diagnostic restriction digestion on an agarose geland further confirmed by DNA sequencing. Plasmid vectors encodinganti-Flt1-EBP diblock blocks were prepared using BseRI, and ligation andconfirmation of ligation were performed as described above.

Example 6: Expression of Genes Encoding EBPs,Anti-Flt1-EBPP[A₁G₄I₁]_(3n) and Anti-Flt1-EBP Diblock Block andPurification of Gene Expression Products

E. coli strain BL21 (DE3) cells were transformed with each vectorcontaining an EBP, anti-Flt1-EBPP[A₁G₄I₁]_(3n) or an anti-Flt1-EBPdiblock block, and then inoculated in 50 ml of CircleGrow mediasupplemented with 50 μg/ml ampicillin Preculture was performed in ashaking incubator at 200 rpm overnight at 37° C. 500 ml of CircleGrowmedia with 50 μg/ml ampicillin was then inoculated with 50 ml of theprecultured CircleGrow media and incubated in a shaking incubator at 200rpm for 16 hours at 37° C. When optical density at 600 nm (OD₆₀₀)reached 1.0, overexpression of an EBP gene or a block polypeptide genethereof was induced by addition of IPTG at a final concentration of 1mM. The cells were centrifuged at 4500 rpm for 10 minutes at 4° C. Theexpressed EBPs and block polypeptides thereof were purified by inversetransition cycling (ITC) as reported previously. The cell pellet wasresuspended in 30 ml of HEPES buffer, and the cells were lysed bysonication for 10 s in 20 s intervals (VC-505, Sonics & Materials, Inc,Danbury, Conn.) on ice. The cell lysate was centrifuged in a 50 mlcentrifuge tube at 13,000 rpm for 15 min at 4° C. to precipitate theinsoluble debris of the cell lysate. Supernatant containing soluble EBPswas then transferred to a new 50 ml centrifuge tube and centrifuged with0.5% w/v of PEI at 13,000 rpm for 15 minutes at 4° C. to precipitatenucleic acid contaminants. The inverse phase transition of the EBPs weretriggered by adding sodium chloride at a final concentration of 4 M, andaggregated EBPs were separated from the lysate solution bycentrifugation at 13,000 rpm for 15 minutes at 4° C. The aggregated EBPswere resuspended in cold PBS buffer, and the EBP solutions werecentrifuged at 13,000 rpm for 15 minutes at 4° C. to remove anyaggregated protein contaminants. These aggregation and resuspensionprocesses were repeated 5 to 10 times until EBP purity reached about95%, and the purity was determined by SDS-polyacrylamide gelelectrophoresis (SDS-PAGE).

FIG. 5A to 5D show a schematic diagram of molecular design, cloning andthe anti-neovascularization function of fusion polypeptides according tothe present invention. As shown in FIG. 5C, a fusion polypeptidecorresponding to VEGFR-targeting peptide (anti-Flt1 peptide)-hydrophilicEBP-hydrophobic EBP forms a temperature-triggered core-shell micellarstructure with a multivalent VEGFR-targeting peptide under physiologicalconditions.

As shown in FIG. 5D (i), a fusion polypeptide corresponding toVEGFR-targeting peptide (anti-Flt1 peptide)-hydrophilic EBP may act as atherapeutic polypeptide due to strong non-covalent interactions betweenVEGFRs (in particular, VEGFR1) and the anti-Flt1 peptide. As shown inFIG. 5D (ii), a fusion polypeptide corresponding to VEGFR-targetingpeptide (anti-Flt1 peptide)-hydrophilic EBP-hydrophobic EBP forms amicelle with a multivalent anti-Flt1 peptide, which increases thebinding affinity of the fusion polypeptide for VEGFRs. Thus, use of thefusion polypeptide may enhance therapeutic efficacy for diseasesassociated with neovascularization. To minimize rapid degradation ofanti-Flt1 peptides and to present anti-Flt1 peptides, as in vivoreceptor antagonists, EBPs were introduced to an anti-Flt1 peptide asnon-chromatographic purification polypeptide tags and as stabilizers.

Modified pET-21a (mpET-21a) plasmids harboring EBPP[A₁G₄I₁]_(n),EBPP[E₁G₄I₁]_(n) or EBPP[G₁A₃F₂]_(n) (where the subscript number n of[X_(i)Y_(j)Z_(k)]_(n) is 6, 12, 18, 24, 30 or 36) were seamlessly clonedusing standard molecular biology methodology. In particular,multimerization and fusion of EBPP genes were executed using recursivedirectional ligation (RDL) to construct genes encoding EBPPs withdifferent molecular weights and EBPP block copolymers. FIG. 5A shows onemethod of gene cloning, by which genes for two different EBPPs and genesfor an oligonucleotide cassette encoding an anti-Flt1 peptide werecombined to prepare a gene for anti-Flt1-EBPP[E₁G₄I₁]₁₂-[G₁A₃F₂]₂₄. AnmpET-21a plasmid harboring EBPP[G₁A₃F₂]₂₄ was double-digested with XbaIand BseRI and dephosphorylated to prepare a linearized vector, whereasan mpET-21a plasmid harboring EBPP[E₁G₄I₁]₁₂ was double-digested withXbaI and BseRI to prepare an insert. After ligation, a gene for a EBPPdiblock of EBPP[E₁G₄I₁]₁₂-[G₁A₃F₂]₂₄ was prepared. The cloned gene wasdigested with BseRI, dephosphorylated, and fused with an oligonucleotidecassette encoding an anti-Fil1 peptide to prepareanti-Flt1-EBPP[E₁G₄I₁]₁₂-[G₁A₃F₂]₂₄. Similarly, a series of genes foranti-Flt1-EBPP[A₁G₄I₁]_(3, 6, 12, 24) andanti-Flt1-EBPP[E₁G₄I₁]₁₂-[G₁A₃F₂]_(12, 24) were cloned, and fusionpolypeptides thereof were synthesized from plasmid-borne genes in E.coli, as shown in FIG. 1 (B).

In VEGFR-targeting peptide (anti-Flt1 peptide)-hydrophilic EBP-fusionpolypeptide, anti-Flt1-EBPP[A₁G₄I₁]_(3, 6, 12, 24) is soluble underphysiological conditions and acts as a VEGFR antagonist to compete withVEGF, thereby inhibiting delivery of neovascularization signals to cells(FIG. 5D(i)). In an embodiment of the present invention,EBPP[A₁G₄I₁]_(n) was selected because EBPP[A₁G₄I₁]_(n) of all lengths ishydrophilic at body temperature without any charged amino acid residues,and because EBPP[A₁G₄I₁]_(n) helps to provide an understanding of thecorrelation between EBPP length and binding affinity of an anti-Flt1peptide according to EBPP blocks of four different lengths ofanti-Flt1-EBPP[A₁G₄I₁]_(3, 6, 12, 24). Furthermore, as shown in FIGS. 5c and 5 d (ii), a fusionpolypeptide[anti-Flt1-EBPP[E₁G₄I₁]₁₂-[G₁A₃F₂]_(12, 24)] ofVEGFR-targeting peptide (anti-Flt1 peptide)-hydrophilic EBP-hydrophobicEBP may form a temperature-triggered core-shell micellar structure witha multivalent VEGFR1-targeting peptide because of amphiphilic propertiesof hydrophilic EBPP[E₁G₄I₁]₁₂ and hydrophobic EBPP[G₁A₃F₂]_(12, 24)under physiological conditions. In addition, these properties enhancethe binding affinity of the fusion peptides to VEGFRs and allow thefusion peptides to have high adhesion. In particular, hydrophobicEBPP[G₁A₃F₂]_(12, 24) of two different lengths was used for micelle sizecontrol, and was used to study the effects of micelle size on thebinding affinity of anti-Flt1-EBPP[E₁G₄I₁]₁₂-[G₁A₃F₂]_(12, 24) toVEGFRs.

Example 7: Characterization of EBPs, Anti-Flt1-EBPP[A₁G₄I₁]_(3n) andAnti-Flt1-EBP Diblock Block

The purity of EBPs, anti-Flt1-EBPP[A₁G₄I₁]_(3n) and anti-Flt1-EBPdiblock blocks was determined by SDS-PAGE, and gel permeationchromatography (GPC) with a high-performance liquid chromatography(HPLC) 1260 series instrument (Agilent Technologies, Palo Alto, Calif.,U.S.) using a Shodex GPC OHpak SB-804 HQ column (Showa Denko Co., Tokyo,Japan). Deionized water at 20° C. was used as an eluent at a flow rateof 1 ml/min and the GPC column was maintained at 20° C. Low dispersitypullulan in a range of 5,900 to 200,000 g/mol was used as a standard. Aseries of EBPs, anti-Flt1-EBPP[A₁G₄I₁]_(3n) and anti-Flt1-EBP diblockblocks were analyzed using a refractive index detector (RID) andvariable wavelength detector (VWD) at 280 nm. An effect of temperatureon the inverse phase transition of various EBPs,anti-Flt1-EBPP[A₁G₄I₁]_(3n) and anti-Flt1-EBP diblock blocks at 25 μMconcentration in PBS was determined by measuring OD₃₅₀ using a Cary 100Bio UV/Vis spectrophotometer equipped with a multi-cell thermoelectrictemperature controller (Varian Instruments, Walnut Creek, Calif.)between 10 to 85° C. at a heating rate of 1° C./min Self-assemblybehaviors of anti-Flt1-EBPP[E₁G₄I₁]₁₂-[G₁A₃F₂]₁₂ and anti-Flt1-EBPP[E₁G₄I₁]₁₂-[G₁A₃F₂]₂₄ from soluble unimers into micelles werecharacterized using a temperature-controlled Nano ZS90 (ZEN3690) dynamiclight scattering (DLS) instrument (Malvern instruments, Worcestershire,UK), and the hydrodynamic radius (R_(H)) thereof at 25 μM in PBS wasmeasured in 11 successive runs at each temperature in a temperaturerange from 18 to 50° C. at a heating rate of 1° C./min. In addition,T_(t) thereof is defined as the onset temperature for phase transition,and calculated from each DLS plot.

Genes for fusion polypeptides composed of an anti-Flt1 peptide andhydrophilic EBP blocks with different lengths were constructed bymolecular cloning and the lengths of those genes digested with XbaI andBseRI were confirmed by agarose gel electrophoresis as shown in FIG.6(A). The DNA length of each gene encoding anti-Flt1-EBPP[A₁G₄I₁]₃,anti-Flt1-EBPP[A₁G₄I₁]₆, anti-Flt1-EBPP[A₁G₄I₁]₁₂ oranti-Flt1-EBPP[A₁G₄I₁]₂₄ (354, 624, 1164 or 2244 bp, from left to right)is indicated below the respective gene fragments. Since DNA sequencesdigested by XbaI and BseRI are located outside genes encoding the fusionpolypeptides, the DNA lengths of the genes are 66 base pairs longer thanoriginal gene lengths shown in Table 4. The fusion polypeptides composedof an anti-Flt1 peptide and EBP blocks with different chain lengths wereexpressed in E. coli and purified by ITC, as previously reported for thetemperature-responsive EBPs. A copper-stained SDS-PAGE gel (4 to 20%gradient) shown in FIG. 6(B) shows the following:Anti-Flt1-EBPP[A₁G₄I₁]_(n) (subscript n is 3, 6, 12, or 24) was purifiedto have a homogeneity of at least 95% by an average of five rounds ofITC as characterized by HPLC. Compared to a standard protein migrationdistance, each fusion polypeptide shifted about 20% more thantheoretical molecular weights shown in Table 4, which is in goodagreement with previous studies. The expected molecular weights of thefusion polypeptides are indicated below each band (8.19, 15.27, 29.42and 57.72 kDa, from left to right), and lanes at both ends of theSDS-PAGE gel represent standard protein size markers (7, 15, 24, 35, 40,50, 65, 90, 110, and 150 kDa, from bottom to top).

FIG. 7 shows the thermal transition behaviors of EBP blocks and thethermal transition behaviors of fusion polypeptides composed of ananti-Flt1 peptide and EBP blocks with different chain lengths. Based onthe thermal transition behaviors, the effect of EBP block length, sodiumchloride concentration and anti-Flt1 peptide fusion on transitiontemperature (T_(t)) may be investigated. Turbidity profiles in FIG. 7were obtained by measuring the absorbance of (A to C) 25 μMEBPP[A₁G₄I₁]_(3n) (n: integer) and (D to F) 25 μManti-Flt1-EBPP[A₁G₄I]_(3n) (n: integer) in 10 mM PBS (A and D) and in 10mM PBS supplemented with 1 M sodium chloride (B and E) or 2 M sodiumchloride (C and F) at 350 nm while heating samples at a rate of 1°C./min. T_(t) is defined as the inflection point of each thermal plot inFIG. 7 and summarized in Table 5.

TABLE 5 (a) (b) (c) (d) (e) (f) (g) (h) 0M NaCl N/A N/A N/A 68 N/A N/A67 57 1M NaCl N/A 80 52 42 N/A 57 45 39 2M NaCl N/A 53 34 28 49 34 28 23T₁ of (a) EBPP[A₁G₄I₁]₃, (b) EBPP[A₁G₄I₁]₆, (c) EBPP[A₁G₄I₁]₁₂, (d)EBPP[A₁G₄I₁]₂₄, (e) anti-Flt1-EBPP[A₁G₄I₁]₃, (f)anti-Flt1-EBPP[A₁G₄I₁]₆, (g) anti-Flt1-EBPP[A₁G₄I₁]₁₂ and (h)anti-Flt1-EBPP[A₁G₄I₁]₂₄

T_(t) values in Table 5 are determined by measuring the inflectionpoints of thermal profiles in FIG. 7 . Transition temperature waschanged depending on EBPP[A₁G₄I₁] block length and sodium chlorideconcentration.

In general, EBPP[A₁G₄I₁]_(3n) and anti-Flt1-EBPP [A₁G₄I₁]_(3n) withoutpolar amino acid residues exhibit T_(t) higher than 37° C. underphysiological conditions because Ala, Gly and Ile were introduced to theEBPPs as the guest residue of the repetitive pentapeptide unit of theEBPPs in a ratio of 1:4:1. Anti-Flt1-EBPP[A₁G₄I₁]_(3n) was hydrophilicand VEGFR binding-fusion polypeptides thereof were soluble underphysiological conditions, which allowed the polypeptides to specificallybind to VEGFRs without any steric hindrance. Thus, the fusionpolypeptides of the present invention may act as VEGFR antagonistsagainst VEGF. Furthermore, when the effect of EBPP block length andionic strength on thermal responsiveness was analyzed, as the EBP blocklength of EBPP[A₁G₄I₁]_(3n) and anti-Flt1-EBPP[A₁G₄I₁]_(3n), and sodiumchloride concentration in PBS increased, T_(t) thereof decreased. Inparticular, the T_(t) of anti-Flt1-EBPP[A₁G₄I₁]_(3n) was much lower thanthat of EBPP[A₁G₄I₁]_(3n) because Gly-Asn-Gln-Trp-Phe-Ile (GNQWFI) of ananti-Flt1 peptide sequence for targeting VEGFRs was hydrophobic,resulting in a decrease in the T_(t) of anti-Flt1-EBPP[A₁G₄I₁]_(3n). Forexample, the T_(t) of anti-Flt1-EBPP[A₁G₄I₁]₁₂ and the T_(t) ofanti-Flt1-EBPP[A₁G₄I₁]₂₄ were about 18 and 11° C. lower than those ofEBPP[A₁G₄I₁]₁₂ and EBPP[A₁G₄I₁]₂₄ in PBS, respectively. A T_(t)difference (DT_(t)) between EBPP[A₁G₄I₁]₃ and anti-Flt1-EBPP[A₁G₄I₁]₃was more than 36° C. in PBS with 2 M sodium chloride, whereas DT_(t)between EBPP[A₁G₄I₁]₃ and anti-Flt1-EBPP[A₁G₄I₁]₃ was 23° C. in PBS with1 M sodium chloride. Therefore, as EBPP[A₁G₄I₁] block length becameshorter, the T_(t) of anti-Flt1-EBPP[A₁G₄I₁]_(3n) was greatly decreasedirrespective of various concentrations of sodium chloride. This dataindicates that the effect of hydrophobicity of the anti-Flt1 peptide onthe thermal transition of the EBPP[A₁G₄I₁] block is potentially greater.

Next, the properties of fusion polypeptides composed of VEGFR-targetingpeptide (anti-Flt1 peptide)-hydrophilic EBP-hydrophobic EBP aredescribed. Two different genes, which encode a fusion polypeptidecomposed of “anti-Flt1 peptide” and “amphiphilic EBP diblock” ofhydrophilic EBP-hydrophobic EBP having hydrophobic EBP blocks withvarious chain lengths, were constructed using RDL, a seamless molecularcloning method. The full lengths of those genes digested by XbaI andBseRI were confirmed by agarose gel electrophoresis as shown in FIG.8(A). The DNA length of each gene encoding (a)anti-Flt1-EBPP[E₁G₄I₁]₁₂-[G₁A₃F₂]₁₂ or (b)anti-Flt1-EBPP[E₁G₄I₁]₁₂-[G₁A₃F₂]₂₄ (2244 and 3324 bp, from left toright) is indicated below each gene fragment. Since DNA sequencesdigested by XbaI and BseRI are located outside genes encoding the fusionpolypeptides, the lengths of these genes are 66 base pairs longer thanthe original gene lengths of the fusion polypeptides the shown in Table4. Two different anti-Flt1-EBP diblock copolypeptides including (a)anti-Flt1-EBPP[E₁G₄I₁]₁₂-[G₁A₃F₂]₁₂ and (b)anti-Flt1-EBPP[E₁G₄I₁]₁₂-[G₁A₃F₂]₂₄ were expressed in E. coli andpurified by one among non-chromatographic purification methods, ITC asdescribed above for purification of a series of temperature-responsiveanti-Flt1-EBPP[A₁G₄I₁]_(3n). The image of a copper-stained SDS-PAGE gel(4 to 20% gradient) in FIG. 8(B) shows the following: Bothanti-Flt1-EBPP[E₁G₄I₁]₁₂-[G₁A₃F₂]₁₂ andanti-Flt1-EBPP[E₁G₄I₁]₁₂-[G₁A₃F₂]₂₄ were purified by an average of fiverounds of ITC, each with one major polypeptide band. Compared to astandard protein migration distance, each fusion polypeptide shiftedabout 20% more than theoretical molecular weights shown in Table 4. Inaddition, as characterized by HPLC, after an average of five rounds ofITC runs, each polypeptide had a homogeneity of at least 95%. Theexpected molecular weights of the polypeptides are indicated below eachband (59.9 and 90.0 kDa, from left to right), and lanes at both ends ofthe SDS-PAGE gel represent standard protein size markers (7, 15, 24, 35,40, 50, 65, 90, 110, and 150 kDa, from bottom to top).

FIG. 9 shows the thermal transition behaviors of anti-Flt1-EBP diblockcopolypeptides of anti-Flt1-EBPP[E₁G₄I₁]₁₂-[G₁A₃F₂]₁₂ andanti-Flt1-EBPP[E₁G₄I₁]₁₂-[G₁A₃F₂]₂₄ depending on the length andconcentration of a hydrophobic EBPP[G₁A₃F₂] block. Turbidity profileswere obtained by measuring absorbance at 350 nm at four differentconcentrations (12.5, 25, 50 and 100 μM) in 10 mM PBS at a heating rateof 1° C./min. As described above, T_(t) was measured as the inflectionpoint of each thermal plot in FIG. 9 and summarized in Table 6 below.

TABLE 6 (a) (b) (c) (d) Conc. (uM) 25 25 12.5 25 50 100 12.5 25 50 100First T_(t) 39.02 29.12 34.2 36.2 37.4 39.5 26.7 28.0 29.1 29.2 (° C.)Second T_(t) N/A N/A 78.2 81.2 82.3 84.4 74.6 78.0 79.2 81.2 (° C.) T₁of (a) EBPP[E₁G₄I₁]₁₂-EBPP[G₁A₃F₂]₁₂, (b) EBPP[E₁G₄I₁]₁₂-EBPP[G₁A₃F₂]₂₄,(c) anti-Flt1-EBPP[E₁G₄I₁]₁₂-EBPP[G₁A₃F₂]₁₂ and (d)anti-Flt1-EBPP[E₁G₄I₁]₁₂-EBPP[G₁A₃F₂]₂₄

T_(t) values in Table 6 are determined by measuring the inflectionpoints of thermal profiles in FIG. 7 . The first phase transitionoccurred as a result of hydrophobic block aggregation, and was greatlyaffected by the length of EBPP[A₁G₃F₂]. The fusion polypeptides thereofhad the same polar EBPP[E₁G₄I₁]₁₂ block. The second phase transition wasaffected by a polar EBPP[E₁G₄I₁]₁₂ block, and the fusion polypeptidesthereof had a similar second T_(t)

As the concentration of anti-Flt1-EBP diblock blocks increased, thefirst T_(t) and the second T_(t) gradually decreased. In general, thetemperature-triggered phase transition of anti-Flt1-EBP diblockcopolypeptides occurs twice, because aliphatic- and hydrophobicEBPP[A₁G₃F₂] block having a low T_(t) and polar- and hydrophilicEBPP[E₁G₄I₁] block having a high T_(t) exhibit different thermalproperties. The phase transition of anti-Flt1-EBPP[E₁G₄I₁]₁₂-[G₁A₃F₂]₁₂occurred at 36.2 and 81.2° C. at the 25 μM concentration, whereas thephase transition of anti-Flt1-EBPP[E₁G₄I₁]₁₂-[G₁A₃F₂]₂₄ occurred at 28.0and 78.0° C. at the same concentration. This data indicates that thedoubled block length of the hydrophobic EBPP[A₁G₃F₂] has a significanteffect on the first T_(t) and the second T_(t), lowering the same by 8.2and 3.2° C., respectively. In particular, diblock polypeptides ofEBPP[E₁G₄I₁]₁₂-[G₁A₃F₂]₁₂ and EBPP[E₁G₄I₁]₁₂-[G₁A₃F₂]₂₄ withoutanti-Flt1 fusion, as a control, exhibited a first T_(t) of only 39.0 and29.1° C. without an additional phase transition, as shown in FIG. 9(A).On the other hand, anti-Flt1-EBP diblock copolypeptides clearlyexhibited a lowered first T_(t) and second T_(t) as opposed to the phasetransition behavior of diblock polypeptides without anti-Flt1, becausefusion of a hydrophobic anti-Flt1 peptide (Gly-Asn-Gln-Trp-Phe-Ile(GNQWFI)) and the hydrophilic EBPP[E₁G₄I₁] block of diblock polypeptidesgreatly decreased the first T_(t) of EBPP[G₁A₃F₂] and the second T_(t)of a hydrophilic EBPP[E₁G₄I₁] block, which was due to the proximity ofthese blocks. Furthermore, in anti-Flt1-EBP diblock copolypeptides,EBPP[A₁G₃F₂] and EBPP[E₁G₄I₁], with block lengths adjusted at exactly1:1 and 1:2 ratios, created a unique metastable micelle phase rightabove the first T_(t) thereof, which indicated that thethermally-triggered amphiphilic anti-Flt1-EBP diblock copolypeptidesself-assembled into a metastable micelle. At this time, the metastablemicelle continued to develop as a stable micelle in a temperature rangefrom the first T_(t) to the second T_(t). This is in good agreement withthe self-assembly behaviors of diblock polypeptides ofEBPP[E₁G₄I₁]₁₂-[G₁A₃F₂]₁₂ and EBPP[E₁G₄I₁]₁₂-[G₁A₃F₂]₂₄ that are notfused with anti-Flt1, as in a EBP-based diblock copolymer-based micellereported previously.

In accordance with the unique thermal transition of anti-Flt1-EBPdiblock copolypeptides, the self-assembly behaviors ofanti-Flt1-EBPP[E₁G₄I₁]₁₂-[G₁A₃F₂]₁₂ andanti-Flt1-EBPP[E₁G₄I₁]₁₂-[G₁A₃F₂]₂₄ from soluble unimers into micelleswere characterized by dynamic light scattering (DLS). The hydrodynamicradius (R_(H)) thereof at 25 μM in PBS was measured in 11 successiveruns at each temperature in a temperature range from 18 to 50° C. at aheating rate of 1° C./min. T_(t) thereof was defined as the onsettemperature for phase transition, calculated from each DLS plot in FIG.10 , and summarized in Table 7 below.

TABLE 7 (a) (b) First T_(t) (° C.) Second T_(t) (° C.) First T_(t) (°C.) Second T_(t) (° C.) Absorbance 36.2 82.3 28.0 79.2 DLS 36.0 N/A 27.0N/A T_(t) of (a) anti-Flt1-EBPP[E₁G₄I₁]₁₂-EBPP[G₁A₃F₂]₁₂ and (b)anti-Flt1-EBPP[E₁G₄I₁]₁₂-EBPP[G₁A₃F₂]₂₄

Referring to Table 7, the first aggregation of fusion polypeptidesincreases the hydrodynamic radius thereof due to micelle formation.

Anti-Flt1-EBP diblock copolypeptides existed in soluble unimer formsbelow the first T_(t) of 36° C. for anti-Flt1-EBPP[E₁G₄I₁]₁₂-[G₁A₃F₂]₁₂and below the first T_(t) of 27° C. foranti-Flt1-EBPP[E₁G₄I₁]₁₂-[G₁A₃F₂]₂₄, and the hydrodynamic radius (R_(H))thereof at 25 μM in PBS was about 10 nm. As temperature increased abovethe first T_(t), the R_(H) thereof instantaneously increased in a rangeof 160 and 240 nm at a slightly higher temperature than the first T_(t),then decreased to 28.4 and 43.6 nm. The anti-Flt1-EBP diblockcopolypeptides formed metastable micelles due to non-equilibriumthermodynamics of amphiphile-based self-assembly and differenthydrophilic-to-hydrophobic block length ratios, and then thecopolypeptides formed stable micelles with constant R_(H) values even at50° C. because self-assembly thereof reached equilibrium. The R_(H) ofan anti-Flt1-EBPP[E₁G₄I₁]₁₂-EBPP[G₁A₃F₂]₂₄ micelle was 15.2 nm largerthan that of the anti-Flt1-EBPP[E₁G₄I₁]₁₂-EBPP[G₁A₃F₂]₁₂ micelle due tothe doubled block length of EBPP[G₁A₃F₂] and the bigger aggregateddomain of the EBPP[G₁A₃F₂] block at the core of the micellar structurethereof. Furthermore, to determine the critical micelle concentrations(CMCs) of the anti-Flt1-EBP diblock copolypeptides, the micelle sizesthereof at various concentrations in a range of 0.1 to 25 μM weremeasured at 20° C. below T_(t) and 37° C. above T_(t). Underenvironmental conditions of 0.5 μM and 37° C., theanti-Flt1-EBPP[E₁G₄I₁]₁₂-[G₁A₃F₂]₁₂ still formed a metastable micellewith a R_(H) of ˜125 nm and the anti-Flt1-EBPP[E₁G₄I₁]₁₂-EBPP[G₁A₃F₂]₂₄formed a stabilized micelle with a R_(H) of ˜44 nm. However, no micelleformation was observed for the two anti-Flt1-EBP diblock copolypeptidesat 0.1 μM and 37° C., indicating that the 0.1 μM concentration was lowerthan CMCs thereof, and the CMCs were in a range of 0.1 to 0.5 μM.Therefore, the anti-Flt1-EBP diblock copolypeptides formedtemperature-triggered core-corona micellar structures with multivalentanti-Flt1 peptides for targeting Flt1 under physiological conditionsbecause of the amphiphilic properties of hydrophilic EBPP[E₁G₄I₁] andhydrophobic EBPP[G₁A₃F₂]. In particular, in the anti-Flt1-EBP diblockcopolypeptides, different block lengths of hydrophobic EBPP[G₁A₃F₂]finely controlled micellar size, which affected the binding affinitythereof to Flt1, resulting in high adhesion.

Example 8: Determination of Specific Binding ofAnti-Flt1-EBPP[A₁G₄I₁]_(3n) and Anti-Flt1-EBP Diblock Block to Flt1

Specific binding of anti-Flt1-EBPP[A₁G₄I₁]_(3n) (n: 1, 2, 4, and 8) andanti-Flt1-EBP diblock copolypeptides to Flt1 was determined byenzyme-linked immunosorbent assay (ELISA). First, to coat a 96 wellplate with recombinant human VEGF165 protein (rhVEGF₁₆₅) (M.W. 38.4 kDa)present in a disulfide-linked homodimer, 50 μl of a solution containingthe rhVEGF₁₆₅ at a concentration of 0.5 μg/ml was added to the 96 wellplate, and the plated was incubated at 4° C. overnight. The wells of the96 well plate coated with the rhVEGF₁₆₅ were washed with PBS containing0.05% Tween-20 to completely remove unattached rhVEGF₁₆₅, and then thewells were incubated with PBS containing 3 wt % BSA at room temperaturefor 2 hours to block the surface of each well, which was not coated withthe rhVEGF₁₆₅. After incubation, the wells were washed with PBScontaining 0.05% Tween-20 to remove unbound BSA. Next, to impartspecific binding affinity between an anti-Flt1 peptide and Flt1(VEGFR1), a recombinant human Flt1-F_(c) chimeric protein (M.W. 200.0kDa) present in a disulfide-linked homodimer at a concentration of 0.5μg/ml was pre-incubated with (1) anti-Flt1-EBPP[A₁G₄I₁]_(3n) (n: 1, 2,4, and 8) in PBS containing 1 wt % BSA or with (2) anti-Flt1-EBP diblockcopolypeptides (anti-Flt1-EBPP[E₁G₄I₁]₁₂-EBPP [G₁A₃F₂] 12 andanti-Flt1-EBPP[E₁G₄I₁]₁₂-EBPP[G₁A₃F₂]₂₄) with hydrophobic blocks ofdifferent lengths. In this case, the pre-incubation was carried out atroom temperature for 2 hours at different concentrations within a rangeof 0.5 to 500 μM. Thereafter, the mixed solution was added torhVEGF₁₆₅-coated wells, followed by additional incubation at roomtemperature for 2 hours. The EBPP[A₁G₄I₁]₁₂ and EBP diblockcopolypeptide (EBPP[E₁G₄I₁]₁₂-EBPP[G₁A₃F₂]₁₂ andanti-Flt1-EBPP[E₁G₄I₁]₁₂-EBPP[G₁A₃F₂]₂₄) with hydrophobic blocks (havingthe same concentration) of different lengths were used as a standard.Each well was washed with PBS supplemented with 0.05% Tween-20 to removeFlt1-F_(c) protein that was not bound to rhVEGF₁₆₅ on the surface of thewell. Whether human Flt1-F_(c) protein was specifically bound to therhVEGF₁₆₅-coated well was determined by measuring the absorbance ofoxidized chromogenic substrates upon protein-antibody binding at 450 nmusing rabbit anti-human IgG F_(c)-horseradish peroxidase (HRP)conjugates as a secondary antibody. PBS (containing 0.3 w % BSA) dilutedwith anti-human IgG F_(c)-HRP was added to each well and incubated for 1hour at room temperature, followed by washing 8 times with PBScontaining 0.05 Tween-20. 3,3′,5,5′-tetramethylbenzidine (TMB) was addedto each well to indirectly determine the degree of specific binding ofFlt1-F_(c) protein to VEGF by measuring the specific interaction betweenthe Flt1-F_(c) protein and the anti-human IgG F_(c)-HRP protein, andHRP-catalyzed oxidation of the TMB. The color intensity of the oxidizedTMB was measured at 450 nm. Each ELISA experiment was performed threetimes for reproducibility.

The specific binding properties of a fusion polypeptide ofVEGFR-targeting peptide (anti-Flt1 peptide)-hydrophilic EBP areexamined. As shown in FIG. 11 , the specific binding ofanti-Flt1-EBPP[A₁G₄I₁]_(3n) (n: integer) fusion polypeptides wascharacterized by enzyme-linked immunosorbent assay (ELISA). First, 38.4kDa recombinant human VEGF₁₆₅ protein present in a disulfide-linkedhomodimer was coated on wells, and then the wells were blocked by bovineserum albumin (BSA). A 200.0 kDa recombinant human Flt1-F_(c) chimericprotein present in a disulfide-linked homodimer was incubated withanti-Flt1-EBPP[A₁G₄I₁]_(3n) (n: 1, 2, 4, and 8) at differentconcentrations within a range of 0.5 to 500 μM to induce specificbinding between each other, and then the mixed solution was added to theVEGF-coated wells, followed by incubation for 2 hours at roomtemperature. Human Flt1-F_(c) chimeric protein was specifically bound tothe VEGF-coated wells was determined by measuring the absorbance ofoxidized chromogenic substrates upon protein-antibody binding at 450 nmusing rabbit anti-human IgG F_(c)-horseradish peroxidase (HRP)conjugates as a secondary antibody. Regardless of differentconcentrations, EBPP[A₁G₄I₁]₁₂ did not significantly inhibit specificbinding between the Flt1-F_(c) chimeric protein and VEGF. Contrary tothe minimal inhibitory effect of EBPP[A₁G₄I₁]₁₂ with respect to thespecific binding, anti-Flt1-EBPP[A₁G₄I₁]_(3n) copolypeptidessignificantly inhibited an interaction between Flt1-F_(c) and VEGF in adose-dependent manner independent of EBPP[A₁G₄I₁] block length. Theseresults indicate that anti-Flt1-EBPP[A₁G₄I₁]_(3n) has a high specificbinding capacity to a human Flt1-F_(c) chimeric protein, which mayprevent the human Flt1-F_(c) chimeric protein from binding to VEGF. Inparticular, anti-Flt1-EBPP[A₁G₄I₁]₁₂ showed a maximum inhibitory effectof about 75% at 500 μM, whereas anti-Flt1-EBPP [A₁G₄I₁]₂₄ had a lowerinhibitory effect than anti-Flt1-EBPP[A₁G₄I₁]₁₂, which might be theconsequence of steric hindrance caused by an extended EBPP[A₁G₄I₁] chainlength. Although hydrophilic EBPP[A₁G₄I₁]₃ blocks with different chainlengths were introduced to anti-Flt1 peptides as VEGFR1-specificantagonists, anti-Flt1-EBPP[A₁G₄I₁]_(3n) copolypeptides retained highspecificity of the anti-Flt1 peptide for Flt1, due to the inert natureof EBPs. In contrast to conventional peptide-polymer conjugates such asanti-Flt1 peptide-hyaluronate (HA) conjugates, theanti-Flt1-EBPP[A₁G₄I₁]_(3n) copolypeptides were prepared at the genelevel, and imparted the monodisperse molecular weight and enhancedstabilization of an anti-Flt1 peptide due to the inert nature of EBPsacting like PEG This monodisperse molecular weight and stability mightincrease the half-life of the anti-Flt1 peptide in vivo.

Next, the binding properties of fusion polypeptides of VEGFR-targetingpeptide (anti-Flt1 peptide)-hydrophilic EBP-hydrophobic EBP are examinedWith specific binding of soluble anti-Flt1-EBPP[A₁G₄I₁]_(3n) to a humanFlt1-F_(c) chimeric protein, anti-Flt1-EBP diblock blocks(anti-Flt1-EBPP [E₁G₄I₁]₁₂-EBPP[G₁A₃F₂]₁₂ andanti-Flt1-EBPP[E₁G₄I₁]₁₂-EBPP [G₁A₃F₂]₂₄) with hydrophobic blocks ofdifferent lengths formed temperature-triggered core-shell micellarstructures with multivalent anti-Flt1 peptides under physiologicalconditions. Multivalent anti-Flt1 located on the outer shell of theformed self-assembled micelles increased the binding affinity of thefusion polypeptides to human Flt1 (VEGFR1). As measured by enzyme-linkedimmunosorbent assay (ELISA) in FIG. 12 , as the concentrations of fusionpolypeptides of anti-Flt1 peptide-hydrophilic EBP-hydrophobic EBPincreased, specific binding between Flt1-F_(c) and VEGF wassignificantly inhibited by the fusion polypeptides, which is in goodagreement with the results of the example for solubleanti-Flt1-EBPP[A₁G₄I₁]_(3n). Unlike the degree of inhibition ofanti-Flt1-EBPP[A₁G₄I₁]_(3n) fusion polypeptides with respect to specificbinding between Flt1-F_(c) and VEGF,anti-Flt1-EBPP[E₁G₄I₁]₁₂-EBPP[G₁A₃F₂]₁₂ micelles with a R_(H) of ˜125 nmin a metastable state showed a dramatically enhanced inhibitory effect(˜95%) on specific binding between Flt1-F_(c) and VEGF depending on thespatial multivalent display of Flt1-targeting peptides on the micelles.On the other hand, anti-Flt1-EBPP[E₁G₄I₁]₁₂-EBPP[G₁A₃F₂]₂₄ micelles witha R_(H) of ˜44 nm in a stable state exhibited a similar inhibitiondegree compared with the degree of inhibition of solubleanti-Flt1-EBPP[A₁G₄I₁]_(3n) blocks. Although the two anti-Flt1-EBPdiblock copolypeptides formed micelles in a concentration range from 0.5to 500 μM at 37° C. under physiological conditions, the peptides showeda much different degree of inhibition with respect to specific bindingbetween Flt1-F_(c) and VEGF based on the stability of micelles thereof,potentially due to different binding affinities between the Flt1-F_(c)and the controlled spatial display of the multivalent anti-Flt1 peptidesof the micellar nanostructures. Importantly,anti-Flt1-EBPP[E₁G₄I₁]₁₂-EBPP[G₁A₃F₂]₁₂ micelles at 250 μM had a greaterinhibitory effect than anti-Flt1-EBPP[A₁G₄I₁]₃ fusion polypeptides at500 μM, which suggested that a lower dose of theanti-Flt1-EBPP[E₁G₄I₁]₁₂-EBPP[G₁A₃F₂]₁₂ might have a higher bindingaffinity to the human Flt1 protein in vivo for anti-neovascularization.

Example 9: In Vitro Tubing Assay of HUVECs UsingAnti-Flt1-EBPP[A₁G₄I₁]₁₂

In vitro tubing assay of HUVECs using soluble anti-Flt1-EBPP[A₁G₄I₁]₁₂was performed to evaluate effects of the solubleanti-Flt1-EBPP[A₁G₄I₁]₁₂ copolypeptides on proliferation, migration andtube formation of endothelial cells. For Matrigel coating, 200 μl of 8.7mg/ml Matrigel was added to a 48 well plate and incubated at 37° C. for1 hour to become solidified. To label HUVECs with fluorescence, HUVECswere incubated with 10 μM calcein-AM at 37° C. for 15 minutes and washedwith PBS several times. The calcein-labeled HUVECs at 2×10⁴ cells/wellwere grown on the Matrigel-coated wells, and incubated at 37° C. for 4hours with 50 ng/ml recombinant human rhVEGF₁₆₅ andanti-Flt1-EBPP[A₁G₄I₁]₁₂ as a Flt1-specific antagonist at differentconcentrations. After incubation, it was determined whetherproliferation, migration and tube formation of endothelial cells werestimulated. To clarify to what extent anti-Flt1-EBPP[A₁G₄I₁]₁₂ couldinhibit tube formation of HUVECs, the same concentration ofEBPP[A₁G₄I₁]₁₂ was assessed as a control. In addition, Avastin, arecombinant humanized monoclonal antibody (mAb) against VEGF, was usedas another control to compare therapeutic efficacy foranti-neovascularization based on the therapeutic efficacy ofanti-Flt1-EBPP[A₁G₄I₁]₁₂, as a Flt1-specific antagonist. The tubeformation of HUVECs was photographed with Micromanipulator (Olympus,Tokyo, Japan), and quantified by measuring whole tube lengths in threerandom fields per well with Image lab software (Bio-Rad Laboratories,Hercules, Calif., USA). When the tubing assay was performed, the tubeformation of HUVECs incubated in PBS for 4 hours was used as a control.The experiment was repeated three times.

As shown in FIG. 11 , based on enzyme-linked immunosorbent assay (ELISA)results, it was confirmed that anti-Flt1-EBPP[A₁G₄I₁]_(3n) (n: integer)fusion polypeptides had a high specific binding capacity to the humanFlt1-F_(c) chimeric protein, and 29.4 kDa anti-Flt1-EBPP[A₁G₄I₁]₁₂showed a maximum degree of inhibition compared with solubleanti-Flt1-EBPP[A₁G₄I₁]_(3n) fusion polypeptides with different EBP chainlengths. Accordingly, the effects of soluble anti-Flt1-EBPP[A₁G₄I₁]₁₂fusion polypeptides on proliferation, migration and tube formation ofendothelial cells were assessed in HUVECs in vitro. Calcein-labeledHUVECs at 2×10⁴ cells/well were grown on a 48 well plate pre-coated withMatrigel, 50 ng/ml recombinant human rhVEGF₁₆₅ was treated to stimulateproliferation, migration and tube formation of endothelial cells, andanti-Flt1-EBPP[A₁G₄I₁]₁₂ acting as a Flt1 (VEGFR1)-specific antagonistwas treated at different concentrations, followed by incubation. Thesame concentration of EBPP[A₁G₄I₁]₁₂ was used as a control to clearlyshow to what extent anti-Flt1-EBPP[A₁G₄I₁]₁₂ could inhibit the tubeformation of HUVECs. In addition, Avastin (also named bevacizumab), arecombinant humanized monoclonal antibody (mAb) against VEGF, was usedas a control to compare therapeutic efficacy foranti-neovascularization. Avastin has been widely used to treat variousneovascular eye diseases, such as age-related macular degeneration (AMD)and diabetic retinopathy, based on specific binding between Avastin andVEGF. Based on specific binding of anti-Flt1-EBPP[A₁G₄I₁]₃ to the humanFlt1 protein in the membrane of HUVECs, it was assumed that theinhibitory effects of anti-Flt1-EBPP[A₁G₄I₁]_(3n) and Avastin on thetube formation of HUVECs were caused by specific protein-proteininteractions, while Avastin bound to rhVEGF₁₆₅ and minimizedrhVEGF₁₆₅-triggered cellular signaling for the tube formation andneovascularization of HUVECs. As characterized by in vitro tubing assayof anti-Flt1-EBPP[A₁G₄I₁]₁₂, fluorescence images of calcein-AM labeledHUVECs and the degree of inhibition of tube formation based on thenormalized tube length of HUVECs in the images were shown in FIG. 13 .The tube length of HUVECs was measured by tracking the fluorescencesignal of HUVECs, averaged under each condition, and the tube length ofHUVECs incubated in PBS for 4 hours was used as a baseline. A tubelength when HUVECs were treated with rhVEGF₁₆₅ for 4 hours was set at100%, and tube length at each concentration was normalized. The tubelength of HUVECs incubated with EBPP[A₁G₄I₁]₁₂ as a control was similarto that of HUVECs treated with rhVEGF₁₆₅, indicating that EBPP[A₁G₄I₁]₁₂had no significant effect on the tube formation of HUVECs. On the otherhand, in the case of anti-Flt1-EBPP[A₁G₄I₁]₁₂ as a Flt1-specificantagonist, as the concentration of the anti-Flt1-EBPP[A₁G₄I₁]₁₂increased in a range of 0.1 to 10 μM, the tube length of HUVECsgradually decreased. In accordance with decrease of the tube length ofHUVECs when incubated with anti-Flt1-EBPP[A₁G₄I₁]₁₂, fluorescence imagesclearly show that the degree of inhibition of migration and tubeformation of HUVECs became evident as the concentration ofanti-Flt1-EBPP[A₁G₄I₁]₁₂ increased. These results indicate thatanti-Flt-EBPP[A₁G₄I₁]₁₂ inhibits the tube formation of HUVECs and thedegree of inhibition is greatly controlled by the concentration ofanti-Flt1-EBPP[A₁G₄I₁]₁₂. In particular, HUVECs incubated with 10 μManti-Flt1-EBPP[A₁G₄I₁]₁₂ showed no significant migration and tubeformation even in the presence of rhVEGF₁₆₅, which was similar to use ofAvastin at 0.2 mg/ml. Therefore, as validated by ELISA, theanti-Flt1-EBPP[A₁G₄I₁]₁₂ fusion polypeptides still retained highspecificity of an anti-Flt1 peptide against Flt1.

Example 10: In Vivo Anti-Neovascularization UsingAnti-Flt1-EBPP[A₁G₄I₁]₁₂ in Laser-Induced Choroidal NeovascularizationModel

6- to 8-week-old female C57BL-6 mice were anesthetized withintraperitoneal injection of ketamine at 100 mg/kg and xylazine at 10mg/kg, and the pupils were dilated with 5 mg/ml tropicamide, and 532 nmlaser diode (150 to 210 mW, 0.1 sec, 50 to 100 μM) was applied to eachfundus to induce choroidal neovascularization in vivo. Multiple burnswere performed in the 6, 9, 12, and 3 o'clock positions of the posteriorpole of the eye with a slit-lamp delivery system. Production of bubblesat the time of laser, which indicates Bruch's membrane rupturing, is animportant factor in obtaining the CNV model. To evaluate an effect ofanti-Flt1-EBPP[A₁G₄I₁]_(3n) copolypeptides on anti-neovascularization ina laser-induced choroidal neovascularization model in vivo, the CNVmodel mice were injected in an intravitreal manner with PBS as avehicle, EBPP[A₁G₄I₁]₁₂ or various concentrations ofanti-Flt1-EBPP[A₁G₄I₁]₁₂ once a day for 5 days and anesthetized after 14days with an intraperitoneal injection of ketamine at 100 mg/kg andxylazine at 10 mg/kg. The mice were treated with retro-orbital injectionof 100 μl ultrapure water containing 25 mg/ml FITC-dextran. Enucleatedeyes were then fixed in 10% formalin for 30 minutes at room temperature.The cornea, iris, lens, and vitreous humor were gently removed under astereomicroscope (Leica, Wetzlar, Germany). Four radial incisions weremade in the dissected retina, which was then flattened with a coverslip.Each in vivo anti-neovascularization experiment was performed with threereplicates.

By ELISA and HUVEC tubing assay, it was demonstrated thatanti-Flt1-EBPP[A G₄I₁]_(3n) fusion polypeptides retainedanti-neovascularization activity as an antagonist against VEGFR1. Thepresent inventors hypothesized that anti-Flt1-EBPP[A₁G₄I₁]_(3n) fusionpolypeptides might show a therapeutic activity with respect toneovascularization-related eye diseases (in particular, retinalneovascular disease, age-related macular degeneration (AMD)).Intravitreal injection of anti-Flt1-EBPP[A₁G₄I₁]₁₂ was evaluated for thesuppression of laser-induced choroidal neovascularization (CNV), whichwas an animal model for AMD, in C57BL-6 mice. Daily injection of proteinsolutions started immediately after laser injury and maintained for 5days. Injection of a vehicle (PBS) or EBPP[A₁G₄I₁]₁₂ was used as anegative control. CNV lesion volumes were imagined and evaluated withfluorescein isothiocyanate (FITC)-dextran perfused whole choroidalflat-mounts at day 14 after laser injury (FIG. 14A). Quantitativeanalysis showed that doses of 0.1, 1, 5 and 20 μg ofanti-Flt1-EBPP[A₁G₄I₁]₁₂ per day for 5 days (in total 0.5, 5, 25 and 100μg) suppressed CNV lesion size by 32%, 52.3% (P<0.05), 54.4% (P<0.05),and 25.9%, respectively, as compared with PBS control mice (FIG. 14B).The CNV lesion sizes of an EBPP[A₁G₄I₁]₁₂-treated animal had valuessimilar to those of a PBS-treated animal. The suppressive effect ofEBPP[A₁G₄I₁]₁₂ on a CNV lesion showed a dose dependent manner in a rangefrom 0.5 to 25 μg in total. However, 100 μg anti-Flt1-EBPP[A₁G₄I₁]₁₂showed a reduced effect on suppression of the CNV lesion, potentiallydue to an excessive dose of anti-Flt1-EBPP[A₁G₄I₁]₁₂.

Binding affinity of a targeting ligand against a growth factor receptor(GFR) in cells is important for various diseases associated with cellgrowth such as neovascularization, because the binding affinitydetermines whether intracellular signaling will proceed. In the presentinvention, VEGFR-targeting fusion polypeptides, which are composed ofthermally responsive elastin-based polypeptides (EBPs) and vascularendothelial growth factor receptor (VEGFR)-targeting peptides, weregenetically manipulated, expressed, and purified and the physicochemicalproperties thereof were analyzed. The EBPs were introduced asnon-chromatographic purification tags and also introduced as astabilizer, like a poly(ethylene glycol) conjugate, for minimizing rapidin vivo degradation of VEGFR-targeting peptides. In addition, theVEGFR-targeting peptide was introduced to function as a receptorantagonist by specifically binding to VEGFRs.

A fusion polypeptide composed of VEGFR-targeting peptide (anti-Flt1peptide)-hydrophilic EBP exhibited a soluble unimer form. On the otherhand, a fusion polypeptide composed of VEGFR-targeting peptide(anti-Flt1 peptide)-hydrophilic EBP-hydrophobic EBP exhibited atemperature-triggered core-shell micellar structure with a multivalentVGFR-targeting peptide under physiological conditions. As analyzed byenzyme-linked immunosorbent assay (ELISA), this structure greatlyincreased the binding affinity of the fusion polypeptide for VEGFreceptors. Depending on the spatial display of a VEGFR-targetingpeptide, the binding affinity of the fusion polypeptides to VEGFRs wasgreatly regulated.

An anti-Flt1-EBPP[A₁G₄I₁]_(3n) fusion polypeptide (anti-Flt1peptide-hydrophilic EBP), which existed as a soluble unimer form below atransition temperature, showed a high anti-neovascularization effect ina CNV model as compared with a EBPP block as a control. In addition, ananti-Flt1-EBP diblock fusion polypeptide (anti-Flt1 peptide-hydrophilicEBP-hydrophobic EBP) formed a temperature-triggered, self-assembledmultivalent micellar nanostructure under physiological conditions,resulting in a great difference in the degree of inhibition with respectto specific binding between Flt1-F_(c) and VEGF depending on thestability of the micellar nanostructure thereof. In the tube formationassay of HUVECs in vitro, anti-Flt1-EBPP[A₁G₄I₁]₁₂ greatly reduced tubeformation, whereas EBPP[A₁G₄I₁]₁₂ had no significant effect on tubeformation, which was due to specific interactions between theanti-Flt1-EBPP[A₁G₄I₁]₁₂ and Flt1 (VEGFR1) on the HUVEC membrane.Finally, in the laser-induced CNV model of mice,anti-Flt1-EBPP[A₁G₄I₁]₁₂ showed a high anti-neovascularization effect.Therefore, this fusion polypeptide and the self-assembled multivalentmicellar nanostructure thereof with an anti-Flt1 may be used as atherapeutic polypeptide targeting neovascularization, such as treatmentof retinal, corneal, choroidal neovascularization, tumor growth, cancermetastasis, diabetic retinopathy, and asthma.

A fusion polypeptide for inhibiting neovascularization of the presentinvention can provide a new direction for a drug delivery system foranti-neovascularization, such as treatment of retinal, corneal,choroidal neovascularization, tumor growth, cancer metastasis, diabeticretinopathy, and asthma.

What is claimed is:
 1. A fusion polypeptide for inhibitingneovascularization, comprising: a peptide of SEQ ID NO: 38 specificallybinding to vascular endothelial growth factor (VEGF) receptors; and ahydrophilic elastin-based polypeptide (hydrophilic EBP) linked to thepeptide, wherein the fusion polypeptide further comprises a hydrophobicelastin-based polypeptide (hydrophobic EBP) linked to the hydrophilicEBP, and the hydrophobic EBP is consisting of an amino acid sequencerepresented by Formula 1 or 2 below: Formula 1 [SEQ ID NO: 1] n; or  Formula 2 [SEQ ID NO: 2] n, wherein   SEQ ID NO: 1 is consisting of[VPGXG VPGXG VPGXG VPGXG VPGXG VPGXG];   SEQ ID NO: 2 is consisting of[VPAXG VPAXG VPAXG VPAXG VPAXG VPAXG];

n is an integer of 1 or more, and represents the number of repeats ofSEQ ID NO: 1 or SEQ ID NO: 2; and X is an amino acid other than proline,is selected from any natural or artificial amino acids when thepentapeptide VPGXG or VPAXG is repeated, and at least one of X is ahydrophobic or aliphatic amino acid.
 2. The fusion polypeptide accordingto claim 1, wherein the hydrophobic EBP is consisting of an amino acidsequence represented by Formula 1 or 2: in Formula 1, n is 1, and each Xof the pentapeptide repeats is consisting of G (Gly), A (Ala), and F(Phe) in a ratio of 1:3:2 [SEQ ID NO: 28], or in Formula 2, n is 1, andeach X of the pentapeptide repeats is consisting of, G (Gly), A (Ala),and F (Phe) in a ratio of 1:3:2 [SEQ ID NO: 29]; K (Lys), A (Ala), and F(Phe) in a ratio of 1:3:2 [SEQ ID NO: 30]; D (Asp), A (Ala), and F (Phe)in a ratio of 1:3:2 [SEQ ID NO: 31]; K (Lys) and F (Phe) in a ratio of3:3 [SEQ ID NO: 32]; D (Asp) and F (Phe) in a ratio of 3:3 [SEQ ID NO:33]; H (His), A (Ala), and I (Ile) in a ratio of 3:2:1 [SEQ ID NO: 34];H (His) and G (Gly) in a ratio of 5:1 [SEQ ID NO. 35]; or G (Gly), C(Cys), and F (Phe) in a ratio of 1:3:2 [SEQ ID NO: 36].
 3. The fusionpolypeptide according to claim 1, wherein the hydrophobic EBP isconsisting of an amino acid sequence represented by Formula 2: inFormula 2, n is 12, and each X of the pentapeptide repeats is consistingof G (Gly), A (Ala), and F (Phe) in a ratio of 1:3:2 [SEQ ID NO: 46], orin Formula 2, n is 24, and each X of the pentapeptide repeats isconsisting of G (Gly), A (Ala), and F (Phe) in a ratio of 1:3:2 [SEQ IDNO: 47].
 4. The fusion polypeptide according to claim 1, wherein thefusion polypeptide is consisting of an amino acid sequence correspondingto SEQ ID NO: 52 or SEQ ID NO:
 53. 5. The fusion polypeptide accordingto claim 1, wherein the fusion polypeptide forms a self-assemblednanostructure having a core-shell structure, when the hydrophobic EBPforms a core structure and the hydrophilic EBP and the VEGFreceptor-specific peptide form a shell structure by a temperaturestimulus ranging from 18 to 50° C.
 6. The fusion polypeptide accordingto claim 5, wherein the self-assembled nanostructure comprises amultivalent VEGF receptor-specific peptide as a shell.
 7. A compositionfor treating diseases caused by neovascularization, comprising thefusion polypeptide of claim 1, wherein the fusion polypeptide forms aself-assembled nanostructure having a core-shell structure, when ahydrophobic EBP forms a core structure and a hydrophilic EBP and a VEGFreceptor-specific peptide form a shell structure by a temperaturestimulus ranging from 18 to 50° C., and the self-assembled nanostructurecomprises a multivalent VEGF receptor-specific peptide as a shell,whereby binding affinity between the self-assembled nanostructure and aVEGF receptor increases, and VEGF fails to bind to the VEGF receptor,thereby inhibiting neovascularization.
 8. The composition according toclaim 7, wherein the diseases caused by neovascularization is any one ormore selected from the group comprising diabetic retinopathy,retinopathy of prematurity, macular degeneration, choroidalneovascularization, neovascular glaucoma, eye diseases caused by cornealneovascularization, corneal transplant rejection, corneal edema, cornealopacity, cancer, hemangioma, hemangiofibroma, rheumatoid arthritis, andpsoriasis.